Search

The aims of this study were to investigate the plasmid-mediated colistin resistance genes ( mcr-1 , mcr-2 , mcr-3 , mcr-4 , and mcr-5 ), phenotypic colistin resistance in Escherichia coli O157:H7 + /H7 - strains isolated from cattle and sheep, and whole-genome sequence (WGS) analysis of colistin-resistant sorbitol fermentative E. coli O157:H7. According to the results, 5 of the 49 isolates were found to harbor mcr-2 and/or mcr-3 genes. Three isolates, including a sorbitol fermentative E. coli O157:H7, were found phenotypically resistant to colistin with a minimum inhibitory concentration value of 128 μg/mL. The genome of sorbitol fermentative E. coli O157:H7 did not show 100% similarity to any of the other genome sequences found in the universal genome database. It has also been determined that this isolate carried 62 different antimicrobial resistance genes. This is the first report of plasmid-mediated mcr-2 and mcr-3 genes carrying E. coli O157:H7 from cattle and sheep isolates and WGS of a colistin-resistant sorbitol fermentative E. coli O157:H7. Findings of this study indicate that cattle and sheep can be an important source of colistin resistance in E. coli O157:H7, and slaughterhouse wastewater might be a significant route for dissemination of the plasmid-mediated colistin genes. Therefore, the use of colistin in veterinary medicine should be restricted to reduce the development of resistance. Also it may be necessary to review the non-sorbitol fermentation-based isolation protocol for not missing the sorbitol fermentative E. coli O157:H7 in epidemiological studies.

This study determined the prevalence, pulsed-field gel electrophoresis profiles, and antimicrobial resistance profile of Listeria monocytogenes, Salmonella enterica, and Escherichia coli O157:H7 isolates from dairy cattle farms in Jordan. Samples from bulk tank milk (n = 305), cattle feces (n = 610), and rectoanal mucosal swabs (n = 610) were collected from 61 dairy cattle farms. We confirmed 32 L. monocytogenes, 28 S. enterica, and 24 E. coli O157:H7 isolates from the samples. The farm-level prevalence (at least 1 positive sample per farm) of L. monocytogenes, S. enterica, and E. coli O157:H7 was 27.9, 19.7, and 23.0%, respectively. The prevalence of L. monocytogenes, S. enterica, and E. coli O157:H7 in bulk tank milk was 7.5, 1.6, and 3.3%, respectively. The prevalence of L. monocytogenes and S. enterica in fecal samples was 1.5 and 3.8%, respectively, and the prevalence of E. coli O157:H7 in rectoanal mucosal swabs was 2.3%. Based on disk diffusion testing, all L. monocytogenes, S. enterica, and E. coli O157:H7 isolates exhibited resistance to at least 1 antimicrobial class. Multidrug resistance (resistance to 3 or more classes of antimicrobials) was exhibited by 96.9% of L. monocytogenes, 91.7% of E. coli O157:H7, and 82.1% of S. enterica isolates. Moreover, 93.8, 79.2, and 57.1% of the L. monocytogenes, E. coli O157:H7, and S. enterica isolates, respectively, were resistant to 5 or more antimicrobial classes. More than 50% of L. monocytogenes isolates were resistant to ampicillin, clindamycin, penicillin, erythromycin, quinupristin-dalfopristin, streptomycin, teicoplanin, linezolid, vancomycin, kanamycin, and tetracycline. More than 50% of S. enterica and E. coli O157:H7 isolates were resistant to ampicillin, cephalothin, nalidixic acid, kanamycin, streptomycin, amoxicillin-clavulanic acid, and tetracycline. The prevalence of the studied pathogens this study was comparable to reports from other countries. The isolated pathogens exhibited a high degree of antimicrobial resistance, suggesting that the bacterial flora of dairy cattle in Jordan are under intense antimicrobial selection pressure. Additional research is required to determine the causes and drivers of resistance, and to develop approaches to mitigating antimicrobial resistance., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The gastrointestinal tract (GIT) of healthy cattle is the main reservoir of enterohaemorrhagic Escherichia coli (EHEC). Therefore, it is crucial to better understand the physiology of EHEC in the bovine GIT. In this study, we demonstrate that aspartate present in bovine small intestine content (BSIC), was exhausted after incubation of the reference EHEC strain EDL933 but was poorly assimilated by the endogenous microbiota. Furthermore, the bovine commensal E. coli strain BG1 appeared less efficient than EDL933 in aspartate assimilation suggesting a competitive ability of EHEC to assimilate this amino acid. Our results strongly suggest that aspartate, internalized via the DcuA aspartate: succinate antiporting system, is then converted to fumarate and carbamoyl-aspartate, the precursor for UMP biosynthesis. Aspartate assimilation by these two pathways conferred a competitive growth advantage to EHEC in BSIC. In summary, supply of intracellular fumarate due to aspartate deamination and used as an electron acceptor for anaerobic fumarate respiration, as well as de novo synthesis of pyrimidine from aspartate appear to be important pathways favouring EHEC persistence in the bovine gut. Aspartate probably represents an ecological niche for EHEC in the bovine small intestine., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

A sampling method that represents a greater proportion of the beef trimmings in a 900-kg combo bin should improve the current pathogen sampling and detection programs used by fresh beef processors. This study compared two novel, nondestructive sampling methodologies (a continuous sampling device [CSD] and a manual sampling device [MSD]) with the current industry methodologies, the N60 Excision (the "gold standard") and N60 Plus, for collection of trim samples. Depending on the experiment, samples were analyzed for naturally occurring Escherichia coli O157:H7 or Salmonella, inoculated surrogates, or indicator organisms in multiple plants, on multiple days, across multiple lean percentage mixtures. Experiments 1A and 1B with natural contamination found no E. coli O157:H7 but similar ( P > 0.05) prevalence of Salmonella (CSD 9.2% versus N60 Excision 6.0%) and similar ( P > 0.05) levels of indicator organisms for CSD compared with both N60 methodologies. In experiments 2 and 3, CSD cloth sampling had the same or higher prevalence of naturally occurring E. coli O157:H7 and E. coli O157:H7 surrogate organisms, as well as similar levels of indicator organisms compared with the N60 methodologies. In experiment 4, MSD cloth sampling detected similar ( P > 0.05) prevalence of E. coli O157:H7 surrogate organisms, as well as slightly lower ( P < 0.05) levels of indicator organisms compared with N60 Plus. In experiment 5, the MSD found similar ( P > 0.05) prevalence of naturally occurring E. coli O157:H7 and the same or slightly higher ( P < 0.05) levels of naturally occurring indicator organisms compared with N60 Plus. In experiment 6, the MSD detected the same ( P > 0.05) prevalence of naturally occurring Salmonella as did N60 Excision. The results of these experiments collectively demonstrate that sampling beef trim using either the CSD or MSD provides organism recovery that is similar to or better than the N60 Excision or the N60 Plus methodologies.

The effect of transportation and lairage on the faecal shedding and post-slaughter contamination of carcasses with Escherichia coli O157 and O26 in young calves (4-7-day-old) was assessed in a cohort study at a regional calf-processing plant in the North Island of New Zealand, following 60 calves as cohorts from six dairy farms to slaughter. Multiple samples from each animal at pre-slaughter (recto-anal mucosal swab) and carcass at post-slaughter (sponge swab) were collected and screened using real-time PCR and culture isolation methods for the presence of E. coli O157 and O26 (Shiga toxin-producing E. coli (STEC) and non-STEC). Genotype analysis of E. coli O157 and O26 isolates provided little evidence of faecal-oral transmission of infection between calves during transportation and lairage. Increased cross-contamination of hides and carcasses with E. coli O157 and O26 between co-transported calves was confirmed at pre-hide removal and post-evisceration stages but not at pre-boning (at the end of dressing prior to chilling), indicating that good hygiene practices and application of an approved intervention effectively controlled carcass contamination. This study was the first of its kind to assess the impact of transportation and lairage on the faecal carriage and post-harvest contamination of carcasses with E. coli O157 and O26 in very young calves.

Enterohemorrhagic Escherichia coli O157:H7 colonizes the gastrointestinal tract of ruminants, including cattle and bison, which are reservoirs of these zoonotic disease-causing bacteria. Healthy animals colonized by E. coli O157:H7 do not experience clinical symptoms of the disease induced by E. coli O157:H7 infections in humans; however, a variety of host immunological factors may play a role in the amount and frequency of fecal shedding of E. coli O157:H7 by ruminant reservoirs. How gastrointestinal colonization by E. coli O157:H7 impacts these host animal immunological factors is unknown. Here, various isogenic mutant strains of a foodborne isolate of E. coli O157:H7 were used to evaluate bacterial killing capacity of macrophages of cattle and bison, the two ruminant species. Cattle macrophages demonstrated an enhanced ability to phagocytose and kill E. coli O157:H7 compared to bison macrophages, and killing ability was impacted by E. coli O157:H7 virulence gene expression. These findings suggest that the macrophage responses to E. coli O157:H7 might play a role in the variations observed in E. coli O157:H7 fecal shedding by ruminants in nature., (Published by Elsevier Ltd.)

On 27 April 2015, Washington health authorities identified Escherichia coli O157:H7 infections associated with dairy education school field trips held in a barn 20-24 April. Investigation objectives were to determine the magnitude of the outbreak, identify the source of infection, prevent secondary illness transmission and develop recommendations to prevent future outbreaks. Case-finding, hypothesis generating interviews, environmental site visits and a case-control study were conducted. Parents and children were interviewed regarding event activities. Odds ratios (OR) and 95% confidence intervals (CI) were computed. Environmental testing was conducted in the barn; isolates were compared to patient isolates using pulsed-field gel electrophoresis (PFGE). Sixty people were ill, 11 (18%) were hospitalised and six (10%) developed haemolytic uremic syndrome. Ill people ranged in age from <1 year to 47 years (median: 7), and 20 (33%) were female. Twenty-seven case-patients and 88 controls were enrolled in the case-control study. Among first-grade students, handwashing (i.e. soap and water, or hand sanitiser) before lunch was protective (adjusted OR 0.13; 95% CI 0.02-0.88, P = 0.04). Barn samples yielded E. coli O157:H7 with PFGE patterns indistinguishable from patient isolates. This investigation provided epidemiological, laboratory and environmental evidence for a large outbreak of E. coli O157:H7 infections from exposure to a contaminated barn. The investigation highlights the often overlooked risk of infection through exposure to animal environments as well as the importance of handwashing for disease prevention. Increased education and encouragement of infection prevention measures, such as handwashing, can prevent illness.

Shiga toxin-producing Escherichia coli O157:H7 (O157) are zoonotic foodborne pathogens and of major public health concern that cause considerable intestinal and extra-intestinal illnesses in humans. O157 colonize the recto-anal junction (RAJ) of asymptomatic cattle who shed the bacterium into the environment through fecal matter. A small subset of cattle, termed super-shedders (SS), excrete O157 at a rate (≥ 104 CFU/g of feces) that is several orders of magnitude greater than other colonized cattle and play a major role in the prevalence and transmission of O157. To better understand microbial factors contributing to super-shedding we have recently sequenced two SS isolates, SS17 (GenBank accession no. CP008805) and SS52 (GenBank accession no. CP010304) and shown that SS isolates display a distinctive strongly adherent phenotype on bovine rectal squamous epithelial cells. Here we present a detailed comparative genomics analysis of SS17 and SS52 with other previously characterized O157 strains (EC4115, EDL933, Sakai, TW14359). The results highlight specific polymorphisms and genomic features shared amongst SS strains, and reveal several SNPs that are shared amongst SS isolates, including in genes involved in motility, adherence, and metabolism. Finally, our analyses reveal distinctive patterns of distribution of phage-associated genes amongst the two SS and other isolates. Together, the results of our comparative genomics studies suggest that while SS17 and SS52 share genomic features with other lineage I/II isolates, they likely have distinct recent evolutionary histories. Future comparative and functional genomic studies are needed to decipher the precise molecular basis for super shedding in O157.

Two experiments were conducted to study the effects of monensin dose on growth performance and O157:H7 shedding in finishing beef cattle. In Exp. 1, 198 heifers (298 ± 1.1 kg BW) were allocated to 1 of 2 treatments consisting of 1) 200 mg/heifer daily of monensin and 2) 400 mg/heifer daily of monensin and fed for 151 d. In Exp. 2, 199 steers (430 ± 1.9 kg BW) were stratified by BW and allocated to 1 of 2 treatments consisting of 1) 0 mg/steer daily of monensin and 2) 400 mg/steer daily of monensin and fed for 128 d. For both experiments, there were 4 pen replicates per treatment. For Exp. 1 and Exp. 2, the model included the fixed effect of treatment for growth performance measures and the fixed effects of treatment, time, and treatment × time interaction, respectively, for O157:H7 shedding. In Exp. 1, final BW was 1.9% greater for heifers fed 400 mg/d monensin than for heifers fed 200 mg/d monensin ( = 0.05). Furthermore, ADG was 4.9% greater ( = 0.05) and G:F was 3.1% greater ( = 0.04) when the heifers were fed 400 mg/d monensin vs. 200 mg/d monensin. Pen prevalence for O157:H7 ( = 0.96) and the percentage of animals in the pen shedding O157:H7 at enumerable levels ( = 0.82) did not differ between heifers fed 200 mg/d monensin and heifers fed 400 mg/d monensin over the 4 sampling periods. For Exp. 2, steers fed the supplement containing monensin had a 1.9% greater final BW ( = 0.04) and a 5.2% greater ADG ( = 0.02) than steers fed a control supplement without monensin. No differences in DMI or G:F were noted across the treatments ( ≥ 0.14). O157:H7 percentage of enumerable cattle within the pen was greater for the steers fed monensin than the control steers not fed monensin than the control steers not fed monensin ( = 0.02) over the 4 sampling periods. However, the percentage of animals in the pen shedding O157:H7 (prevalence positive) did not differ between treatments ( = 0.18), nor did the average fecal counts ( = 0.45). In conclusion, feeding a higher dose (400 mg/d) of monensin improved final BW and ADG compared with a low dose of monensin or a no-monensin control in steers and heifers across multiple years. The percentage of animals shedding O157:H7 at enumerable levels was greater for steers fed the monensin supplement than for steers fed the control supplement, yet the presence of monensin, irrespective of the dose, did not affect the percentage of animals in the pen shedding O157:H7.

Clinical outcomes of Shiga toxin (stx)-producing Escherichia coli infection are largely determined by virulence gene subtypes. This study used a polymerase chain reaction (PCR)-pyrosequencing assay to analyze single-nucleotide polymorphisms for subtyping three major virulence genes (stx 1 , stx 2 , eae) of pathogenic E. coli (O157, O26, O111, and O103) isolated from cattle over a 2-year interval (n = 465) and human clinical cases (n = 42) in western Canada. Most bovine isolates were PCR positive for at least one target virulence gene (367/465), whereas 100% of human isolates harbored eae in combination with at least one stx gene. Four Shiga toxin (1a, 2a, 2c, and 2e) and four eae (λ/γ1-eae, ɛ-eae, θ/γ2-eae, and β-eae) subtypes were identified in over 25 distinct virulence genotypes. Among cattle isolates, every serogroup, but O103, presented a dominant genotype (O157: stx 1a +stx 2a +λ/γ1-eae, O26: β-eae alone, and O111: stx 1a +θ/γ2-eae). Similar patterns were found in human isolates, although it was not possible to establish a clear genotypic association between the two sources. Many O157 and non-O157 cattle isolates lacked stx genes; the absence was greater in non-O157 (75/258) and O157:non-H7 (19/40) than in O157:H7 strains (1/164). In addition, there was a greater diversity of virulence genotypes of E. coli isolated from cattle than those of human diseases, which could be due to sample characteristics (e.g., source and clinical condition). However, the majority of cattle strains had virulence profiles identical to those of clinical cases. Consequently, determining the presence of certain stx (stx 1a and stx 2a ) and eae (λ/γ1-eae) subtypes known to cause human disease would be a valuable tool for risk assessment and prediction of disease outcome along the farm-to-fork continuum.

Meat can be contaminated in different stages of the slaughtering process and the identification of these stages is the starting point to implement adequate control measures. The objectives of this study were to assess the presence of pathogenic microorganisms in cattle carcasses, to identify the most important contamination points of the slaughtering process, and to evaluate the possible risk factors related to them in a cattle slaughterhouse. To this aim, 108 cattle carcasses were sampled at three stages of the slaughtering process: Point 1 (hides after bleeding); Point 2 (carcasses after hide removal); and Point 3 (carcasses immediately after division). Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Livingstone were isolated from the carcasses. Phenotypic and genotypic characterization indicated that there was cross-contamination among animals, since bacteria with identical genotypic and phenotypic profiles were isolated from different animals at the same sampling day. Furthermore, this is the first report about the isolation of E. coli O157:H7 in a bovine slaughterhouse from southern Brazil., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study aims to describe in detail the temporal dynamics of E. coli O157 shedding and risk factors for shedding in a grass-fed beef herd. During a 9-month period, 23 beef cows were sampled twice a week (58 sampling points) and E. coli O157 was enumerated from faecal samples. Isolates were screened by PCR for presence of rfbE, stx 1 and stx 2 . The prevalence per sampling day ranged from 0% to 57%. This study demonstrates that many members of the herd were concurrently shedding E. coli O157. Occurrence of rainfall (P < 0·01), feeding silage (P < 0·01) and lactating (P < 0·01) were found to be predictors of shedding. Moving cattle to a new paddock had a negative effect on shedding. This approach, based on short-interval sampling, confirms the known variability of shedding within a herd and highlights that high shedding events are rare.

Cattle are an important reservoir for the foodborne pathogens Salmonella and Escherichia coli O157:H7; they frequently harbor these microorganisms in their digestive tracts and shed them in their feces. Thus, there is potential for contamination of cattle hides and, subsequently, carcasses. Interventions aimed at reducing or eliminating pathogen shedding preharvest will also reduce the likelihood of beef product contamination by these pathogens. Therefore, this study used an in vitro model to evaluate Bdellovibrio bacteriovorus, a gram-negative microorganism that preys upon other gram-negative microorganisms, as a preharvest intervention to control Salmonella and E. coli O157:H7. Rumen fluid and feces were inoculated with pansusceptible or antimicrobial-resistant strains of one pathogen. Control samples were treated with HEPES buffer, whereas experimental samples were exposed to HEPES buffer plus B. bacteriovorus. Salmonella and E. coli O157:H7 populations were quantified at 0, 24, 48, and 72 h. The most-probable-number (MPN) technique, followed by streaking onto xylose lysine Tergitol 4 agar, was used to determine Salmonella populations, whereas spread plating onto sorbitol MacConkey agar supplemented with cefixime and tellurite was employed to enumerate E. coli O157:H7. B. bacteriovorus reduced pansusceptible Salmonella in cattle feces by 2.02 Log MPN/g (P = 0.0005) and antimicrobial-resistant Salmonella by 3.79 (P < 0.0001) and 2.24 (P = 0.0013) Log MPN/g after 24 and 48 h, respectively, in comparison to control samples. Significant reductions were not observed for E. coli O157:H7 in rumen or feces. These data suggest that further investigation into B. bacteriovorus efficacy as a preharvest intervention to control Salmonella in cattle is warranted.

Escherichia coli O157 is an emergent pathogen associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. Meat products constitute an important transmission source of this microorganism. The aims of this study were to characterize E. coli O157 isolated from cattle and meat products collected from abattoirs and retail stores, to establish the clonal relatedness among regional isolates and to compare them with those in the national database. Between 2004 and 2013, 169 minced meat, 35 sausage and 216 carcass samples were analyzed. Thirteen E. coli O157 isolates were identified; 6 of which were O157:H7 and characterized as stx2c(vh-a)/eae/ehxA (n = 5) and stx2/eae/ehxA (n = 1). The 7 remaining isolates were non-toxigenic E. coli strains, and serotyped as O157:NT (n = 4), O157:NM (n = 1), O157:ND (n = 1) and O157:H16 (n = 1). The strains yielded different XbaI-PFGE patterns. Compared to the E. coli O157 isolates in the National Database, none of these patterns have been previously detected in strains of different origin in Argentina., (Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Shiga toxin-producing Escherichia coli O157:H7 (O157) are significant foodborne pathogens and pose a serious threat to public health worldwide. The major reservoirs of O157 are asymptomatic cattle which harbor the organism in the terminal recto-anal junction (RAJ). Some colonized animals, referred to as "super-shedders" (SS), are known to shed O157 in exceptionally large numbers (>104 CFU/g of feces). Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. Whole genome sequence analysis of an SS O157 strain (SS17) revealed a genome of 5,523,849 bp chromosome with 5,430 open reading frames and two plasmids, pO157 and pSS17, of 94,645 bp and 37,446 bp, respectively. Comparative analyses showed that SS17 is clustered with spinach-associated O157 outbreak strains, and belongs to the lineage I/II, clade 8, D group, and genotype 1, a subgroup of O157 with predicted hyper-virulence. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci. Phenotypic analyses revealed a distinctive and strongly adherent aggregative phenotype of SS17 on bovine RAJ stratified squamous epithelial (RSE) cells that was conserved amongst other SS isolates. Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism. Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.

The ability to maintain a dual lifestyle of colonizing the ruminant gut and surviving in nonhost environments once shed is key to the success of Escherichia coli O157:H7 as a zoonotic pathogen. Both physical and biological conditions encountered by the bacteria are likely to change during the transition between host and nonhost environments. In this study, carbon starvation at suboptimal temperatures in nonhost environments was simulated by starving a New Zealand bovine E. coli O157:H7 isolate in phosphate-buffered saline at 4 and 15°C for 84 days. Recovery of starved cells on media with different nutrient availabilities was monitored under aerobic and anaerobic conditions. We found that the New Zealand bovine E. coli O157:H7 isolate was able to maintain membrane integrity and viability over 84 days and that the level of recovery depended on the nutrient level of the recovery medium as well as the starvation temperature. In addition, a significant difference in carbon utilization was observed between starved and nonstarved cells., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Escherichia coli O157:H7 is a major foodborne human pathogen causing disease worldwide. Cattle are a major reservoir for this pathogen and those that shed E. coli O157:H7 at >104 CFU/g feces have been termed "super-shedders". A rich microbial community inhabits the mammalian intestinal tract, but it is not known if the structure of this community differs between super-shedder cattle and their non-shedding pen mates. We hypothesized that the super-shedder state is a result of an intestinal dysbiosis of the microbial community and that a "normal" microbiota prevents E. coli O157:H7 from reaching super-shedding levels. To address this question, we applied 454 pyrosequencing of bacterial 16S rRNA genes to characterize fecal bacterial communities from 11 super-shedders and 11 contemporary pen mates negative for E. coli O157:H7. The dataset was analyzed by using five independent clustering methods to minimize potential biases and to increase confidence in the results. Our analyses collectively indicated significant variations in microbiome composition between super-shedding and non-shedding cattle. Super-shedders exhibited higher bacterial richness and diversity than non-shedders. Furthermore, seventy-two operational taxonomic units, mostly belonging to Firmicutes and Bacteroidetes phyla, were identified showing differential abundance between these two groups of cattle. The operational taxonomic unit affiliation provides new insight into bacterial populations that are present in feces arising from super-shedders of E. coli O157:H7.

Escherichia coli O157:H7 is a zoonotic human pathogen for which cattle are an important reservoir host. Using both previously published and new sequencing data, a 48-locus single nucleotide polymorphism (SNP)-based typing panel was developed that redundantly identified 11 genogroups that span six of the eight lineages recently described for E. coli O157:H7 (J. L. Bono, T. P. Smith, J. E. Keen, G. P. Harhay, T. G. McDaneld, R. E. Mandrell, W. K. Jung, T. E. Besser, P. Gerner-Smidt, M. Bielaszewska, H. Karch, M. L. Clawson, Mol. Biol. Evol. 29:2047-2062, 2012) and additionally defined subgroups within four of those lineages. This assay was applied to 530 isolates from human and bovine sources. The SNP-based lineage groups were concordant with previously identified E. coli O157:H7 genotypes identified by other methods and were strongly associated with carriage of specific Stx genes. Two SNP lineages (Ia and Vb) were disproportionately represented among cattle isolates, and three others (IIa, Ib, and IIb) were disproportionately represented among human clinical isolates. This 48-plex SNP assay efficiently and economically identifies biologically relevant lineages within E. coli O157:H7.

The distribution of virulence factors (VFs) typical of diarrheagenic Escherichia coli and the antimicrobial resistance (AMR) profiles were assessed in 780 isolates from healthy pigs, broilers, and cattle from Spain. VF distribution was broader than expected, although at low prevalence for most genes, with AMR being linked mainly to host species.

Due to the expense of monitoring multiple serotypes of Escherichia coli at slaughter, a study was conducted at a beef abattoir in southern Alberta to determine relationships between E. coli and E. coli O157:H7 on hides. Swab samples were collected from carcasses immediately prior to hide removal over 8 weeks in summer (n = 591) and winter (n = 686). Detection of E. coli was highest in summer (P < 0.05), although detection of E. coli O157:H7 did not differ by season. Numbers of E. coli did not vary by season, but were affected by slaughter plant hygiene schedules. E. coli O157:H7 was more likely (P < 0.001) to be detected on hides of carcasses with the most E. coli (>3.5 log CFU/50 cm²). For E. coli < 3.5 log CFU/50 cm², the likelihood of detecting E. coli O157:H7 did not differ. Consequently, for 83% of carcasses, there was no relationship between numbers of E. coli and detection of E. coli O157:H7 on hides.

Escherichia coli O157 is a foodborne pathogen that can be transmitted by contaminated ground beef and is shed naturally in cattle feces. Recent reports indicated that feeding distillers' grains (DG) to cattle increased fecal shedding and prevalence of E. coli O157. In Minnesota, feeding DG with solubles (DGS) to livestock became widespread within the last 10 years, but there is no report about the prevalence of E. coli O157 in beef cattle in this state. This study was undertaken to survey the fecal prevalence of E. coli O157 in cattle fed diets containing DG and its association with environmental conditions and management practices. Fecal samples were collected from three feedlots during a 1-year period. All animals in those feedlots were fed different DGS levels. E. coli O157 presence was determined using a combination of enrichment, immunomagnetic separation, plating onto sorbitol MacConkey agar, and confirmation of isolates by immunoassay and multiplex virulence genes polymerase chain reaction analysis. Overall, E. coli O157 was confirmed in 9.7% of samples. Prevalence during summer was 30% and declined to less than 10% the rest of the year. In animals grouped by dietary DGS concentration, no significant difference in prevalence (12.0 and 5.5%) was detected between the low and the high average groups (less and more than 20%). Previous feeding of DGS before arriving to the feedlot also had no influence on fecal prevalence. The presence of several interacting variables, uncontrolled in a real-life feedlot environment, was the likely reason for our observation and suggested that at the levels studied, DGS had no effect on the STEC O157 prevalence in cattle populations.

To determine the prevalence of Salmonella and Escherichia coli O157:H7 in cattle feedlots and the impact of subsequent contamination on carcasses in a Mexican Federal Inspection Type Standards harvest facility, 250 animals were tagged and sampled in each step of the slaughter process. Samples were taken from hides and fecal grabs, and composite samples were taken from three anatomical carcass sites (hindshank, foreshank, and inside round) during the slaughter process, at preevisceration (PE), prior to entering the hot box (PHB), and after 24 h of dry chilling (DC). Additionally, 250 fecal samples were collected from the feedlot (FL), holding pens (HP), and intestinal feces (IF), and water samples were taken from the HP area. E. coli O157:H7 and Salmonella detection were carried out with the BAX System, immunomagnetic separation, and conventional methods. Overall Salmonella prevalence was 52.5%. The highest prevalence (92.4%) was found on hides, followed by feces from the HP (91.0%), FL (55.56%), PE (49.0%), IF (46.8%), and PHB (24.8%), for all sampling periods combined. The lowest prevalence of 6.0% was found after DC. The overall prevalence of E. coli O157:H7 was as follows: 11.7% for hides, 5.2% for IF, 2.7% for FL, 2.0% for HP, 0.8% for PE, 0.4% for PHB, and 0.4% for the cooler. High prevalence of Salmonella in IF and on hides present a significant risk factor for contamination by Salmonella at the different processing steps. These results serve as a warning as to the risks of contamination in meats for these pathogens and the importance of following good manufacturing practices during beef production processes.

While efforts to control foodborne illness associated with the Shiga toxin-producing Escherichia coli (E. coli) O157 through processes and procedures implemented at harvest facilities have been very successful, there is concern about the burden of illness associated with other Shiga toxin-producing E. coli. The U.S. Department of Agriculture Food Safety and Inspection Service announced plans to classify an additional six non-O157 Shiga toxin-producing E. coli as adulterants. Little is known about the prevalence and distribution of these E. coli in the animal production environment. An investigation of the prevalence of O157 and the six major non-O157 E. coli serogroups was conducted in 21 feedlots over the period July 2011 to October 2011. Individual fecal swabs were collected from cattle approximately 60 days after their arrival in the feedlot and were pooled for evaluation using a polymerase chain reaction assay to identify the presence of seven E. coli O-types (O157, O45, O103, O121, O145, O26, and O111) and four virulence genes (stx1, stx2, eaeA, and ehxA). Overall, 1145 fecal pools were evaluated, with 506 (44.2%) being positive for one or more of the E. coli O-serogroups. The pool prevalences for E. coli O157, O45, O26, O103, O121, O145, and O111 were 19.7%, 13.8%, 9.9%, 9.3%, 5.5%, 1.1%, and 0.5%, respectively. Nearly all pools were positive for ehxA (99.7%) or stx2 (98.6%). The pool level prevalence for stx1 and eae was 65.5% and 69.3%, respectively. Pools that were positive for one or more of the other E. coli O-serogroups were 1.37 times more likely to be positive for E. coli O157. Conversely, pools that were positive for E. coli O157 were 1.43 times more likely to be positive for at least one of the other E. coli O-serogroups evaluated. These data will be useful to understand the expected prevalence of potential Shiga toxin-producing E. coli in cattle feedlots.

Tannins from forages grown (n = 10) on the Canadian prairie, as well as from Quebracho, Rhus semialata, and brown seaweed (Ascophyllum nodosum), were screened for anti-Escherichia coli O157:H7 activity against E. coli O157:H7 strain 3081 at a concentration of 400 μg/ml for each tannin type, except for brown seaweed, which was at 50 μg/ml. Growth of the bacteria was assessed by measuring the optical density at 600 nm over 24 h. Tannin from seaweed at a concentration of 50 μg/ml inhibited growth of strain 3081. Among the terrestrial forages, only condensed tannins (CT) from purple prairie clover (Dalea purpurea Vent; PPC) increased (P < 0.05) the lag time and reduced (P < 0.05) the growth rate of E. coli O157:H7. The anti-E. coli O157:H7 activity of PPC CT was further assessed by culturing E. coli strain ATCC 25922 and eight strains of E. coli O157:H7 with PPC CT at 0, 25, 50, 100, or 200 μg/ml. Selected strains were enumerated after 0, 6, and 24 h of incubation, and fatty acid composition was determined after 24 h of incubation. E. coli strain 25922 was cultured with 0, 50, or 200 μg of CT per ml and harvested during the exponential growth phase for examination by transmission electron microscopy. Increasing CT concentration linearly increased (P < 0.001) the lag times of seven strains and linearly reduced (P < 0.001) the growth rates of eight E. coli O157:H7 strains. Proportions of unsaturated fatty acids in the total fatty acids were decreased (P < 0.01) by CT at 50 μg/ml. Transmission electron microscopy showed that CT disrupted the outer membrane structure. Anti-E. coli O157:H7 activity of PPC CT at levels of up to 200 μg/ml was bacteriostatic rather than bactericidal, and the mechanism of anti-E. coli activity may involve alteration in the fatty acid composition and disruption of the outer membrane of the cell.

Two naturally occurring, generally recognized as safe compounds, namely, caprylic acid (CA) (1%) and b -resorcylic acid (BR) (1%), and their combination, applied at 23 and 60°C were evaluated for their antimicrobial effects against Escherichia coli O157:H7 on cattle hides in the presence and absence of bovine feces. Fresh cleaned cattle hides were cut into pieces (5 cm(2)), air dried, and inoculated with a five-strain mixture of nalidixic acid-resistant (50 μg/ml) E. coli O157:H7 (∼8.0 log CFU). The hide samples were air dried under a biosafety hood for 2 h and sprayed with 95% ethanol, 1% CA, 1% BR, or a mixture of 1% CA and 1% BR at 23 or 60°C. The hide samples were kept at 23°C, and E. coli O157:H7 populations were determined at 2 and 5 min after treatment. Both CA and BR were effective in decreasing E. coli O157:H7 populations on hides by 3 to 4 log CFU/cm(2) (P < 0.05). Sterile bovine feces had no effect on the decontaminating property of CA and BR on cattle hides (P > 0.05). Results of this study indicate that CA and BR could potentially be used to decontaminate cattle hides, but follow-up research under slaughterhouse conditions is warranted.

The One Health approach investigates the complexities surrounding the interplay between the animal, human and environmental domains. Zoonotic pathogens, such as Escherichia coli (E. coli) O157, which have animal reservoirs, also have direct impact on public health. Cattle are the primary reservoir of E. coli O157, however infection with this bacterium is asymptomatic. Infected cattle appear healthy and have normal growth rates and milk production abilities. Each year, thousands of Canadians become ill following exposure to E. coli O157. To reduce this public health risk, immunization of cattle would be an effective intervention. Several models have shown that on-farm pathogen reduction programs would significantly reduce the risk of human illness.

The disinfectant and antibiotic susceptibility profiles of 344 Escherichia coli O157:H7 strains from cattle carcasses, feces, and hides and ground beef from the United States were determined. A low prevalence of antibiotic resistance was observed (14%). The highest prevalences of resistance were to sulfisoxazole (10.5%), tetracycline (9.9%), streptomycin (7%), and chloramphenicol (4.9%). Four strains were resistant to eight antibiotics (two strains from ground beef and one strain each from hide and preevisceration carcass swabs of cull cattle at harvest). Pulsed-field gel electrophoresis analysis of the E. coli O157:H7 strains revealed two major groups (designated 1 and 2) composed of 17 and 20 clusters, respectively. Clusters 1A, 1B, 1C, and 1G.1 were associated with multidrug-resistant strains. There was no observed correlation between disinfectant resistance and antibiotic resistance. Sixty-nine (20%) of the 344 strains were resistant to chlorhexidine or benzalkonium chloride or the MICs of benzyldimethyldodecylammonium chloride were elevated. Inducible resistance was observed at elevated concentrations of antibiotics (1.4%) and disinfectants (6.1%). The highest rate of disinfectant inducible resistance was to OdoBan, quaternary ammonium chlorides, and the surface disinfectants F25, FS512, and MG, which are used in dairies, restaurants, and food processing plants. High MICs (1,024 to 4,096 m g/ml) of acetic, lactic, and citric acids were found. The decreasing order of acid potency based on molar MICs (MICs(molar)) was acetic, citric, and lactic acid. The correlation of the concentration of dissociated organic acids and MICs(molar) strongly suggests that the observed inhibition of E. coli O157:H7 was primarily due to dissociated forms of the acids.

Feeding corn dried distillers' grain with solubles (DDGS) has been linked to increased fecal shedding of Escherichia coli O157:H7 in cattle. A study was conducted to compare the impact of three diets containing (dry matter basis) 40% corn DDGS, 40% wheat DDGS, or 20% corn and 20% wheat mixed DDGS to a standard barley grain finishing diet on fecal shedding in cattle challenged with a 10(10) CFU mixture of four nalidixic acid-resistant E. coli O157:H7 strains. Rectal grab samples (n = 544) were collected over 70 days and screened for E. coli O157:H7 by direct plating and immunomagnetic bead separation. Feeding diets containing DDGS had no effect (P > 0.05) on the intensity or duration of fecal shedding of E. coli O157:H7 compared with the standard barley grain finishing diet.

We developed a stochastic simulation model to evaluate the impact of Escherichia coli O157:H7 (O157) vaccination on key epidemiological outcomes. The model evaluated a reduction in the O157 prevalence in feedlot cattle as well as concentration in cattle feces due to vaccination. The impact of this reduction on outcomes at slaughter/harvest and consumption was evaluated by simulating the relationships between the O157 prevalence and concentration at various points in the ground beef supply chain. The uncertainty and variability associated with the O157 contamination was explicitly modeled in production, slaughter, and consumption modules. Our results show that vaccination can have a significant benefit with respect to relevant outcomes such as (1) the number of human O157 illnesses due to the consumption of ground beef, (2) the number of production lots with high O157 contamination levels, (3) the likelihood of detection by U.S. Department of Agriculture Food Safety and Inspection Service testing, and (4) the probability of multiple illnesses due to ground beef servings from the same lot. These results show that these outcomes are strongly impacted by preharvest vaccination. For example, if the vaccine is used so as to reduce the prevalence of E. coli shedding cattle by 80% and if all U.S. steers and heifers were vaccinated, the expected number of human illnesses from ground beef-associated O157 would be reduced almost 60%. If the vaccine is 60% or 40% effective, the illness rate would be reduced approximately 45% or 40%, respectively. The number of production lots (10,000-lb lots) with high O157 contamination levels (> 1000 servings) would be reduced by 96% if all steers and heifers received an 80% effective vaccine regimen. The analysis shows that resulting reduction in the number of shedding animals and the reduced concentration of E. coli on carcasses can combine to reduce human illnesses and cost to beef packers.

Shiga toxin-producing Escherichia coli (STEC) cause nonbloody (NBD) and bloody diarrhea (BD), and hemolytic uremic syndrome (HUS). Cattle have been described as their main reservoir. STEC O157:H7 is recognized as the predominant serotype in clinical infections, but much less is known about the dominant subtypes in humans and animals or their genetic relatedness. The aims of this study were to compare the STEC O157 subtypes found in sporadic human infections with those in the bovine reservoir using stx-genotyping, phage typing, and XbaI-pulsed-field gel electrophoresis (PFGE), and correlate the subtypes with the severity of clinical manifestations. The 280 STEC O157:H7 strains collected included in this study were isolated from HUS (n=122), BD (n=69), and NBD (n=30) cases, and healthy carriers (n=5), and from bovines (n=54) in the abattoirs. The stx-genotyping showed that stx₂/stx(2c(vh-a)) was predominant in human (76.1%) and in bovine strains (55.5%), whereas the second more important genotype was stx₂ (20.8%) in human and stx(2c(vh-a)) (16.7%) in cattle strains. In human strains, PT4 (37.6%), PT49 (24.3%), and PT2 (18.6%) were the most frequent PTs (80.5%). In bovine isolates, PT2 (26%), PT39 (16.7%), and PT4 and PT49 (11.1% each) were predominant. By XbaI-PFGE, all 280 strains yielded 148 patterns with 75% similarity, and 169 strains were grouped in 37 clusters. Identical PT-PFGE-stx profile combinations were detected in strains of both origins: PT4-AREXH01.0011-stx₂/stx(2c(vh-a)) (12 humans and one bovine), PT4-AREXH01.0543-stx₂/stx(2c(vh-a)) (one human and four bovines), PT2-AREXH01.0076-stx₂/stx(2c(vh-a)) (one human and four bovines), PT49-AREXH01.0175-stx₂/stx(2c(vh-a)) (seven humans and one bovine), and PT49-AREXH01.0022-stx₂/stx(2c(vh-a)) (seven humans and one bovine). No correlation was found among the stx-genotypes, the phage type, and the clinical symptoms.

Few hard data are available on emergent diseases. However, the need to mitigate and manage emergent diseases has prompted the use of various expert consultation and opinion elicitation methods. We adapted best-worst scaling (BWS) to elicit experts' assessment of the relative practicality and effectiveness of measures to reduce human exposure to E. coli O157. Cattle vaccination was considered the most effective and hand-washing was considered the most practical measure. BWS proved a powerful tool for expert elicitation as it breaks down a cognitively burdensome process into simple, repeated, tasks. In addition, statistical analysis of the resulting data provides a scaled set of scores for the measures, rather than just a ranking. The use of two criteria (practicality and effectiveness) within the BWS process allows the identification of subsets of measures judged as potentially performing well on both criteria, and conversely those judged to be neither effective nor practical.

The locus of enterocyte effacement (LEE) of Escherichia coli O157:H7 (O157) encodes a type III secretion system (T3SS) for secreting LEE-encoded and non-LEE-encoded virulence proteins that promote the adherence of O157 to intestinal epithelial cells and the persistence of this food-borne human pathogen in bovine intestines. In this study, we compared hha sepB and hha mutants of O157 for LEE transcription, T3SS activity, adherence to HEp-2 cells, persistence in bovine intestines, and the ability to induce changes in the expression of proinflammatory cytokines. LEE transcription was upregulated in the hha sepB and hha mutant strains compared to that in the wild-type strain, but the secretion of virulence proteins in the hha sepB mutant was severely compromised. This reduced secretion resulted in reduced adherence of the hha sepB mutant to Hep-2 cells, correlating with a significantly shorter duration and lower magnitude of fecal shedding in feces of weaned (n = 4 per group) calves inoculated with this mutant strain. The levels of LEE transcription, T3SS activity, and adherence to HEp-2 cells were much lower in the wild-type strain than in the hha mutant, but no significant differences were observed in the duration or the magnitude of fecal shedding in calves inoculated with these strains. Examination of the rectoanal junction (RAJ) tissues from three groups of calves showed no adherent O157 bacteria and similar proinflammatory cytokine gene expression, irrespective of the inoculated strain, with the exception that interleukin-1β was upregulated in calves inoculated with the hha sepB mutant. These results indicate that the T3SS is essential for intestinal colonization and prolonged shedding, but increased secretion of virulence proteins did not enhance the duration and magnitude of fecal shedding of O157 in cattle or have any significant impact on the cytokine gene expression in RAJ tissue compared with that in small intestinal tissue from the same calves.

The purpose of this study was to investigate carriage and transfer of verocytotoxigenic Escherichia coli (VTEC) O157, O26, O111, O103 and O145 from faeces and hide to dressed carcasses of Irish cattle as well as establishing the virulence potential of VTEC carried by these cattle. Individual cattle was tracked and faecal samples, hide and carcass (pre-evisceration and post-wash) swabs were analysed for verotoxin (vt1 and vt2) genes using a duplex real-time PCR assay. Positive samples were screened for the five serogroups of interest by real-time PCR. Isolates were recovered from PCR positive samples using immunomagnetic separation and confirmed by latex agglutination and PCR. Isolates were subject to a virulence screen (vt1, vt2, eaeA and hlyA) by PCR. Isolates carrying vt genes were examined by Pulsed-Field Gel Electrophoresis (PFGE). Of the VTEC isolated, E. coli O157 was the most frequently recovered from hide (17.6%), faeces (2.3%) and pre-evisceration/post-wash carcass (0.7%) samples. VTEC O26 was isolated from 0.2% of hide swabs and 1.5% of faeces samples. VTEC O145 was isolated from 0.7% of faeces samples. VTEC O26 and VTEC O145 were not recovered from carcass swabs. Non-VTEC O103 was recovered from all sample types (27.1% hide, 8.5% faeces, 5.5% pre-evisceration carcass, 2.2% post-wash carcass), with 0.2% of hide swabs and 1.0% of faeces samples found to be positive for VTEC O103 isolates. E. coli O111 was not detected in any samples. For the four serogroups recovered, the direct transfer from hide to carcass was not observed. This study shows that while VTEC O157 are being carried by cattle presented for slaughter in Ireland, a number of other verotoxin producing strains are beginning to emerge., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In the environment, bacteriophages are regarded as natural vector for the transmission of Shiga-toxin genes among Shiga-toxin Escherichia coli strains. The possibility of transduction has been noticed in intestinal tract of various animals but experimental observations on this phenomenon in food processes are lacking. To investigate the transduction in milk at different temperature profiles and cell concentrations, an experimental plan including two different Stx(2)-phages (ϕGV2412 and ϕL34), induced respectively from E. coli O157:H7 181181/2 and E. coli O157:H7 EC34, and two recipient E. coli strains (CNCTC 6896, WG5) was performed. The donor strains were generated by lysogenization of CNCTC 6896 with ϕGV2412 and ϕL34 respectively. Spectinomycin resistance gene (aadA) was inserted into stx(2) operon in order to select transduced cells. Transductants were never observed at 4°C up to 24 h, whereas after a treatment at 37°C for 2 h and at 25°C for 22 h they were detected in 67% of the trials with a ratio of transduction varying from 1.13 10(-6) to 7.87 10(-8). A treatment at 48°C for 2 h followed by a second step at 25°C for 22 h showed an occurrence of transduction events in only 19% of cases with a ratio of transduction varying from 2.22 10(-7) to 2.67 10(-8). The generation of transductants and the spontaneous induction of phages in milk were not affected by initial or final concentration of the donor or recipient strains. The results show that transduction phenomenon occurs when the cells are metabolically active and it does not take place at low temperatures. Therefore, the maintenance of the chilling chain proved to be a main factor to prevent the spread of Stx-genes in dairy processes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Three experiments were conducted to evaluate the influence of vitamin D on fecal shedding of Escherichia coli O157:H7 in cattle. In the first experiment, two groups of cattle (beef and dairy) were assigned to a control treatment or to receive 0.5 × 10(6) IU vitamin D per day via oral bolus for 10 days. Fecal samples were collected before and throughout the dosing period for culture of E. coli O157:H7. No differences were observed for fecal shedding of E. coli O157:H7 among treatments for either beef or dairy animals. Serum concentrations of vitamin D were markedly higher (P < 0.0001) in treated beef cattle but only tended to be higher (P = 0.09) in the dairy cattle. In the second experiment, three successive vitamin D dosages (2,400, 4,800, and 9,600 IU/day; 14 days each) were administered to 14 dairy steers (7 steers served as controls), fecal samples were collected daily, and serum samples were collected weekly throughout the 42-day experimental period. No significant differences in fecal prevalence or serum vitamin D concentrations were observed for any of the vitamin D dosages. A third experiment sampled feedlot cattle (winter and summer) to determine whether serum vitamin D concentrations were correlated with fecal shedding of E. coli O157:H7. A fecal sample and a blood sample were obtained in each season from 60 randomly selected animals (total of 120 fecal samples and 120 corresponding blood samples). As expected, season was highly correlated (r = 0.66) with serum vitamin D concentration with higher concentrations (P < 0.01) observed in the summer. E. coli O157:H7 prevalence (percentage of positive samples) was not highly correlated (r = 0.16) with season, although the correlation tended to be significant (P = 0.08). The proportion of cattle shedding E. coli O157:H7 was 16.7 and 6.7% for the summer and winter collections, respectively. Results of this research do not support a correlation between vitamin D intake and E. coli O157:H7 shedding in cattle.

Feedlot pen soil is a source for transmission of Escherichia coli O157:H7, and therefore a target for preharvest strategies to reduce this pathogen in cattle. The objective of this study was to determine the ability of soil solarization to reduce E. coli O157:H7 in feedlot surface material (FSM). A feedlot pen was identified in which naturally occurring E. coli O157:H7 was prevalent and evenly distributed in the FSM. Forty plots 3 by 3 m were randomly assigned such that five plots of each of the solarization times of 0, 1, 2, 3, 4, 6, 8, and 10 weeks were examined. Temperature loggers were placed 7.5 cm below the surface of each plot, and plots to be solarized were covered with clear 6-mil polyethylene. At each sampling time, five FSM samples were collected from each of five solarized and five unsolarized plots. E. coli concentrations and E. coli O157:H7 presence by immunomagnetic separation and plating were determined for each FSM sample. Initial percentages of E. coli O157:H7-positive samples in control and solarized FSM were 84 and 80%, respectively, and did not differ (P > 0.05). E. coli O157:H7 was no longer detectable by 8 weeks of solarization, but was still detected in unsolarized FSM at 10 weeks. The average initial concentration of E. coli in FSM was 5.56 log CFU/g and did not differ between treatments (P > 0.05). There was a 2.0-log decrease of E. coli after 1 week of solarization, and a >3.0-log reduction of E. coli by week 6 of solarization (P, 0.05). E. coli levels remained unchanged in unsolarized FSM (P > 0.05). Daily peak FSM temperatures were on average 8.7°C higher for solarized FSM compared with unsolarized FSM, and reached temperatures as high as 57°C. Because soil solarization reduces E. coli O157:H7, this technique may be useful for reduction of persistence and transmission of this pathogen in cattle production, in addition to remediation of E. coli O157:H7-contaminated soil used to grow food crops.

The aim of the study was to determine the influence of temperature, i.e. 4 and 20 degrees C on the Escherichia coli O157:H7 survival time in a stored cattle slurry in a laboratory model experiment. The results of this investigation indicated that the tested microorganisms underwent a gradual elimination in the cattle slurry, whereas their inactivation rate was clearly dependent on the temperature. A higher survival rate was found in Escherichia coli O157:H7 at 4 degrees C where a theoretical survival time of these microorganisms, determined using a regression analysis, amounted to 83 days. Our study indicates that there is a necessity for the slurry to undergo hygienization processes and that a constant monitoring of liquid animal excreta in search for pathogenic microorganisms is required.

Aims: To provide molecular epidemiological evidence of avian transmission of Escherichia coli O157:H7 between dairy farms in Ohio, this study was designed to identify genetic relatedness between isolates originating from bovine faecal samples and intestinal contents of European starlings captured on these farms., Methods and Results: During a three-year period (2007-2009), cattle (n = 9000) and starlings (n = 430) on 150 different dairy farms in northern Ohio were sampled for the presence of E. coli O157:H7. Isolates were subjected to multiple-locus variable-nucleotide tandem repeat analysis (MLVA). Distinct allelic groups were identified on most farms; however, isolates clustering into three MLVA groups originated from both cattle and birds on different farms., Conclusions: Sharing of indistinguishable epidemiologically linked E. coli O157 MLVA subtypes between starlings and cattle on different farms supports the hypothesis that these birds contribute to the transmission of E. coli O157:H7 between dairy farms., Significance and Impact of Study: A continued need exists to identify and to improve preharvest measures for controlling E. coli O157:H7. Controlling wildlife intrusion, particularly European starlings, on livestock operations, may be an important strategy for reducing dissemination of E. coli O157:H7 between farms and thereby potentially decreasing the on-farm prevalence of E. coli O157:H7 and enhancing the safety of the food supply., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Cattle fed finishing diets with wet distillers grains with solubles (WDGS) have been shown to harbor increased Escherichia coli O157:H7 populations in the feces and on the hides. To determine if feeding a lower level of WDGS at the end of the feeding period reduces E. coli O157:H7 load at harvest, 608 heifers were sorted into one of five treatments and fed 0, 40, or 70% WDGS (dry matter basis). For three of the treatments, WDGS was reduced midway through the study. Treatment 0W0W heifers (positive control) were fed a corn grain-based diet continuously, and 40W40W heifers (negative control) were fed 40% WDGS continuously. Heifers subjected to treatments 40W0W, 40W15W, and 70W15W were fed either 40 or 70% WDGS for the first 56 days and switched to 0 or 15% WDGS, respectively, for the last 56 days. Prior to the switch in diets, animals fed diets with 40 or 70% had higher prevalence and percent enumerable fecal samples for E. coli O157:H7. After the dietary switch, animals fed 40W0W, 40W15W, and 70W15W diets had fecal prevalence and percent enumerable samples (33.4 and 6.3%, 31.0 and 9.7%, and 34.9 and 8.4%, respectively) similar to those of animals fed 0W0W diets (10.2 and 3.2%, respectively; P > 0.05), whereas animals fed 40W40W had the highest fecal prevalence and percent enumerable samples (70.1 and 29.2%, respectively; P < 0.05). Similar relationships between the treatments were observed for hide samples. Time after dietary switch was important, as animals fed lower levels had significantly lower fecal prevalence and percent enumerable samples after 56 days, but not after 28 days. The study indicates that cattle can be switched to lower levels of dietary WDGS (15% or less) 56 days prior to harvest to significantly reduce E. coli O157:H7 in feces and on hides.

Effects of cattle transportation on hide contamination with Escherichia coli O157 have been variable, and the present study was designed to clarify relationships among duration of transport, microclimate, and environment within the trailer and contamination of hides with E. coli O157. Crossbred Angus heifers from a feedlot in southern Alberta were sorted into 10 replicate loads containing 45 animals (short haul, 621.5 ± 2.1 kg of body weight) or 46 animals (long haul, 576.0 ± 1.7 kg of body weight). Long-haul trips (n = 5) were made in July and August to slaughter plant A, whereas short-haul trips (n = 5) were made in October to slaughter plant B. The same trailer unit and driver were used for all loads. Data loggers were located in the ceiling of each compartment of the trailers to record ambient temperature and relative humidity. Each heifer was swabbed on the perineum on-farm prior to loading and immediately after stunning at the slaughter plant (an average 12-h transport for long haul and 1-h transport for short haul). Swabs were transported on ice before immunomagnetic separation for detection of E. coli O157. Transportation did not affect prevalence of hide contamination with E. coli O157, although 80% of long-haul swabs were positive on-farm compared with 26% of short-haul swabs, due to seasonality of shedding E. coli O157. Cattle transported in the nose compartment had fewer positive hide swabs (P < 0.05) than cattle in the deck or belly compartments during long-haul trips, even though the nose had a higher (P < 0.05) temperature-humidity index than the other compartments. Prevalence of hide contamination varied widely among loads even when the organism was at seasonally low levels. This suggests that the feedlot pen has a greater effect on hide contamination at the slaughter plant than transportation factors including temperature-humidity index, loading density, and duration of transport.

Field studies evaluating the effects of multiple concurrent preharvest interventions for Escherichia coli O157 are logistically and economically challenging; however, modeling techniques may provide useful information on these effects while also identifying crucial information gaps that can guide future research. We constructed a risk assessment model with data obtained from a systematic search of scientific literature. Parameter distributions were incorporated into a stochastic Monte Carlo modeling framework to examine the impacts of different combinations of preharvest and harvest interventions for E. coli O157 on the risk of beef carcass contamination. We estimated the risk of E. coli O157 carcass contamination conditional on preharvest fecal prevalence estimates, inclusion of feed additive(s) in the diet, vaccination for E. coli O157, transport and lairage effects, hide intervention(s), and carcass intervention(s). Prevalence parameters for E. coli O157 were assumed to encompass potential effects of concentration; therefore, concentration effects were not specifically evaluated in this study. Sensitivity analyses revealed that fecal prevalence, fecal-to-hide transfer, hide-to-carcass transfer, and carcass intervention efficacy significantly affected the risk of carcass contamination (correlation coefficients of 0.37, 0.56, 0.58, and -0.29, respectively). The results indicated that combinations of preharvest interventions may be particularly important for supplementing harvest interventions during periods of higher variability in fecal shedding prevalence (i.e., summer). Further assessments of the relationships among fecal prevalence and concentration, hide contamination, and subsequent carcass contamination are needed to further define risks and intervention impacts for E. coli O157 contamination of beef.

Escherichia coli O157:H7 causes foodborne illness in humans; cattle are considered a primary reservoir for the organism, and transmission is often through contaminated food products or water. The objective of this study was to determine the genetic diversity of E. coli O157:H7 within a single individual bovine fecal sample based on pulsed-field gel electrophoresis (PFGE) typing. Fecal samples (n=601) were collected from dairy and beef cattle at three separate facilities, and E. coli O157:H7 was isolated by enrichment, immunomagnetic separation, and plating on selective medium. The prevalence of E. coli O157:H7 was 46 (7.7%) of 601. From each positive fecal sample, up to 10 putative colonies were tested, and isolates from samples with at least seven positive colonies were subtyped using PFGE and tested for six major virulence genes by multiplex PCR. A total of 254 E. coli O157:H7 isolates from 27 samples met these criteria and were included in PFGE analysis. Fifteen PFGE subtypes (<100% Dice similarity) were detected among the 254 isolates, and there were no common subtypes between the three locations. Seven (26%) of 27 fecal samples had E. coli O157:H7 isolates with different PFGE subtypes (mean=2.1) within the same sample. The virulence gene profiles of different isolates from the same sample were always identical, regardless of the number of PFGE types. The results of this study suggest that determining the PFGE pattern of a single isolate from a bovine sample may not be sufficient when comparing isolates from feces, hides, or carcasses, because multiple PFGE subtypes are present., (Copyright ©, International Association for Food Protection)

Rectal content grab samples were collected from 2436 beef cattle reared on 406 beef farms in Japan between November 2007 and March 2008. STEC strains O157 and O26 were isolated from 110 (27.1%) and 7 (1.7%) farms, respectively. Farms that tested positive for STEC O157 were located in 35 out of all 47 Japanese prefectures. This indicates that STEC O157 strains are widespread on beef farms nationwide. Of the 2436 tested beef cattle, 218 (8.9%) and 10 (0.4%) had STEC strains O157 and O26 in the rectal content, respectively. The most common Shiga toxin genes detected in the isolated STEC O157 strains were: stx(2c) alone (32.1%), stx(2)/stx(2c) (27.2%), and stx(1)/stx(2) (21.8%). Almost all of the STEC O157 and STEC O26 strains expressed Shiga toxins (Stx). Most of the STEC O157 and STEC O26 strains possessed eaeA and EHEC-hlyA. These results strongly suggest that STEC strains O157 and O26 from beef cattle would be pathogenic to humans. Therefore, it is important to reduce STEC strains O157 and O26 in beef cattle in order to prevent foodborne disease caused by STEC. The presence of dogs and/or cats on a farm was significantly (P=0.02) associated with the prevalence of STEC O157. More research is needed to clarify the role of dogs and cats., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The objective of this study was to define locations on the carcass with highest contamination of E. coli O157 throughout the harvest process and implement targeted interventions to reduce or eliminate contamination. To establish a pathogen baseline, samples were collected at the foreshank, hindshank, inside round, neck and midline area and evaluated for E. coli O157:H7 presence. Environmental samples were also collected in the harvest area and the fabrication area of the facility. E. coli O157:H7 prevalence was highest on the foreshank, hindshank and inside rounds in the baseline study and steam vacuums/cones were implemented as an intervention in these specific areas on the harvest floor. At pre-evisceration, foreshank prevalence of E. coli O157:H7 was significantly (P<0.05) reduced from 21.7% to 3.1% after the application of steam interventions. At the final rail, foreshank prevalence in the baseline study was 4.2% while no E. coli O157:H7 was detected post-intervention implementation. E. coli O157:H7 on hindshanks and inside rounds was significantly reduced after intervention implementation from 24.2 to 11.5% and 37.5 to 16.7%, respectively at the final rail. Pathogen contamination of environmental samples collected in fabrication declined from 6.7% to 0.7% after slaughter interventions were implemented. Data indicate the identifying areas of contamination on the carcass and implementing interventions can significantly reduce E. coli O157 on the carcasses and in the fabrication environment., (© 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.)

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant human pathogen that resides in healthy cattle. It is thought that a reduction in the prevalence and numbers of EHEC in cattle will reduce the load of EHEC entering the food chain. To this end, an intervention strategy involving the addition of chitosan microparticles (CM) to feed in order to reduce the carriage of this pathogen in cattle was evaluated. Experiments with individual Holstein calves and a crossover study found that the addition of CM to feed decreased E. coli O157:H7 shedding. In the crossover study, CM resulted in statistically significant reductions in the numbers recovered from rectal swab samples (P < 0.05) and the duration of shedding (P < 0.05). The effects of feeding CM to calves differed, indicating that the optimal levels of CM may differ between animals or that other factors are involved in the interaction between CM and E. coli O157:H7. In vitro studies demonstrated that E. coli O157:H7 binds to CM, suggesting that the reduction in shedding may result at least in part from the binding of positively charged CM to negatively charged E. coli cells. Additional studies are needed to determine the impact of CM feeding on animal production, but the results from this study indicate that supplementing feed with CM reduces the shedding of E. coli O157:H7 in cattle.

The bovine gastrointestinal tract is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Characterization of nutrients that promote the carriage of these pathogens by the ruminant would help to develop ecological strategies to reduce their survival in the bovine gastrointestinal tract. In this study, we show for the first time that free ethanolamine (EA) constitutes a nitrogen source for the O157:H7 EHEC strain EDL933 in the bovine intestinal content because of induction of the eut (ethanolamine utilization) gene cluster. In contrast, the eut gene cluster is absent in the genome of most species constituting the mammalian gut microbiota. Furthermore, the eutB gene (encoding a subunit of the enzyme that catalyses the release of ammonia from EA) is poorly expressed in non-pathogenic E. coli. Accordingly, EA is consumed by EHEC but is poorly metabolized by endogenous microbiota of the bovine small intestine, including commensal E. coli. Interestingly, the capacity to utilize EA as a nitrogen source confers a growth advantage to E. coli O157:H7 when the bacteria enter the stationary growth phase. These data demonstrate that EHEC strains take advantage of a nitrogen source that is not consumed by the resident microbiota, and suggest that EA represents an ecological niche favouring EHEC persistence in the bovine intestine.

Aims: To establish whether investigated subtyping methods could identify any specific characteristics that distinguish Swedish VTEC O157:H7 strains isolated from cattle farms associated with human enterohaemorrhagic Escherichia coli (EHEC) cases from cattle strains isolated in prevalence studies., Methods and Results: Strains (n = 32) isolated in a dairy herd prevalence study and strains isolated from farms associated with human cases (n = 13) were subjected to typing. Partial sequencing of the vtx(2) genes could not identify any unique variants of vtx(2) or vtx(2c) in strains associated with human cases. A specific variant of VTEC O157:H7, which was overrepresented among farms associated with human cases (P = 0·01), was by two different single-nucleotide-polymorphism (SNP) assays identified as clade 8, a subgroup of VTEC O157:H7 strains considered to be putatively hypervirulent. Multi-locus variable number tandem repeat analysis (MLVA) typing of all strains produced similar results as pulsed-field gel electrophoresis (PFGE) typing regarding clustering of the strains, but MLVA distinguished slightly better among strains than PFGE., Conclusion: In Sweden, VTEC O157:H7 strains from the putatively hypervirulent clade 8 are overrepresented among isolates from cattle farms associated with human cases compared with VTEC O157:H7 strains isolated in prevalence studies., Significance and Impact of the Study: Real-time PCR SNP typing for clade 8 can be used to identify cattle farms that are at higher risk of causing EHEC infections in humans., (© 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.)

Objective: To evaluate seasonal patterns and risk factors for Escherichia coli O157:H7 in feces in a beef cattle herd and determine strain diversity and transition in E coli over time by use of multiple-locus variable-number tandem-repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE)., Sample Population: 456 samples of freshly passed feces collected over a 1-year period from cattle in a range-based cow-calf operation located in the foothills of the Sierra Nevada Mountains in California., Procedures: E coli O157:H7 was recovered from feces by use of immunomagnetic separation and 2 selective media. Virulence factors were detected via reverse transcriptase-PCR assay. Escherichia coli O157:H7 isolates were subtyped with MLVA and PFGE. Prevalence estimates were calculated and significant risk factors determined. A dendrogram was constructed on the basis of results of MLVA typing., Results: Overall prevalence estimate for E coli O157:H7 was 10.5%, with the prevalence lowest during the winter. Mean temperature during the 30 days before collection of samples was significantly associated with prevalence of E coli O157:H7 in feces. Nineteen MLVA and 12 PFGE types were identified., Conclusions and Clinical Relevance: A seasonal pattern was detected for prevalence of E coli O157:H7 in feces collected from beef cattle in California. Subtyping via MLVA and PFGE revealed a diversity of E coli O157:H7 strains in a cow-calf operation and noteworthy turnover of predominant types. Given the importance of accurately determining sources of contamination in investigations of disease outbreaks in humans, MLVA combined with PFGE should be powerful tools for epidemiologists.