Escherichia coli O26, O45, O103, O111, O121, O145, and O157 are the predominant Shiga toxin–producing E. coli (STEC) serogroups implicated in outbreaks of human foodborne illness worldwide. The increasing prevalence of these pathogens has important public health implications. Beef products have been considered a main source of foodborne human STEC infections. Robust and sensitive methods for the detection and characterization of these pathogens are needed to determine prevalence and incidence of STEC in beef processing facilities and to improve food safety interventions aimed at eliminating STEC from the food supply. This study was conducted to develop Taqman real-time multiplex PCR assays for the screening and rapid detection of the predominant STEC serogroups associated with human illness. Three serogroup-specific assays targeted the O-antigen gene clusters of E. coli O26 (wzy), O103 (wzx), and O145 (wzx) in assay 1, O45 (wzy), O111 (manC), and O121 (wzx) in assay 2, and O157 (rfbE) in assay 3. The uidA gene also was included in the serogroup-specific assays as an E. coli internal amplification control. A fourth assay was developed to target selected virulence genes for Shiga toxin (stx1 and stx2), intimin (eae), and enterohemolysin (ehxA). The specificity of the serogroup and virulence gene assays was assessed by testing 100 and 62 E. coli strains and non–E. coli control strains, respectively. The assays correctly detected the genes in all strains examined, and no cross-reactions were observed, representing 100% specificity. The detection limits of the assays were 103 or 104 CFU/ml for pure cultures and artificially contaminated fecal samples, and after a 6-h enrichment period, the detection limit of the assays was 100 CFU/ml. These results indicate that the four real-time multiplex PCR assays are robust and effective for the rapid and reliable detection of the seven predominant STEC serogroups of major public health concern and the detection of their virulence genes.

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Experimental oral challenge studies with three different genotypes of Escherichia coli O157:H7 were conducted in cattle to determine the genotype-specific variability in shedding frequencies and concentrations and the frequency and extent of contamination of the environment. The results indicated that the E. coli O157:H7 genotype and ecological origin maybe important factors for the occurrence and concentration in the cattle host. Four groups of six young Holstein steers each were orally challenged with 10 6 CFU of one of three E. coli O157:H7 strains: FRIK 47 (groups 1 and 2), FRIK 1641 (group 3), and FRIK 2533 (group 4). Recto-anal mucosal swabs (RAMS) and environmental samples were taken on alternate days over 30 days. The numbers of E. coli O157:H7 cells and generic E. coli cells per sample were determined. Also, the presence and absence of 28 gene targets were determined for 2,411 isolates using high-throughput real-time PCR. Over the study period, strains FRIK 47, FRIK 1641, and FRIK 2533 were detected in 52%, 42%, and 2% of RAMS, respectively. Environmental detection of the challenge strains was found mainly in samples of the hides and pen floors, with strains FRIK 47, FRIK 1641, and FRIK 2533 detected in 22%, 27%, and 0% of environmental samples, respectively. Based on the panel of 28 gene targets, genotypes of enterohemorrhagic E. coli (EHEC) and generic E. coli from the experimental samples were clustered into three subgroups. In conclusion, the results suggested that the type and intensity of measures to control this pathogen at the preharvest level may need to be strain specific.

This study reports analysis of faecal shedding dynamics in cattle for three Escherichia coli O157:H7 (ECO157) strains (S1, S2 and S3) of different genotype and ecological history, using experimental inoculation data. The three strains were compared for their shedding frequency and level of ECO157 in faeces. A multistate Markov chain model was used to compare shedding patterns of S1 and S2. Strains S1 and S2 were detected seven to eight times more often and at 104 larger levels than strain S3. Strains S1 and S2 had similar frequencies and levels of shedding. However, the total time spent in the shedding state during colonization was on average four times longer for S1 (15 days) compared to S2 (4 days). These results indicate that an ECO157 strain effect on the frequency, level, pattern and the duration of faecal shedding may need to be considered in control of ECO157 in the cattle reservoir.

ObjectivesThis study examined how environmental, health, social, behavioural and genetic factors interact to contribute to myocardial infarction (MI) risk.DesignSurvey data collected by Wisconsin Longitudinal Study (WLS), USA, from 1957 to 2011, including 235 environmental, health, social and behavioural factors, and 77 single- nucleotide polymorphisms were analysed for association with MI. To identify associations with MI we utilized recursive partitioning and random forest prior to logistic regression and chi-squared analyses.Participants6198 WLS participants (2938 men; 3260 women) who (1) had a MI before 72 years and (2) had a MI between 65 and 72 years.ResultsIn men, stroke (LR OR: 5.01, 95% CI 3.36 to 7.48), high cholesterol (3.29, 2.59 to 4.18), diabetes (3.24, 2.53 to 4.15) and high blood pressure (2.39, 1.92 to 2.96) were significantly associated with MI up to 72 years of age. For those with high cholesterol, the interaction of smoking and lower alcohol consumption increased prevalence from 23% to 41%, with exposure to dangerous working conditions, a factor not previously linked with MI, further increasing prevalence to 50%. Conversely, MI was reported in <2.5% of men with normal cholesterol and no history of diabetes or depression. Only stroke (4.08, 2.17 to 7.65) and diabetes (2.71, 1.81 to 4.04) by 65 remained significantly associated with MI for men after age 65. For women, diabetes (5.62, 4.08 to 7.75), high blood pressure (3.21, 2.34 to 4.39), high cholesterol (2.03, 1.38 to 3.00) and dissatisfaction with their financial situation (4.00, 1.94 to 8.27) were significantly associated with MI up to 72 years of age. Conversely, often engaging in physical activity alone (0.53, 0.32 to 0.89) or with others (0.34, 0.21 to 0.57) was associated with the largest reduction in odds of MI. Being non-diabetic with normal blood pressure and engaging in physical activity often lowered prevalence of MI to 0.2%. Only diabetes by 65 (4.25, 2.50 to 7.24) and being exposed to dangerous work conditions at 54 (2.24, 1.36 to 3.69) remained significantly associated with MI for women after age 65, while still menstruating at 54 (0.46, 0.23 to 0.91) was associated with reduced odds of MI.ConclusionsTogether these results indicate important differences in factors associated with MI between the sexes, that combinations of factors greatly influence the likelihood of MI, that MI-associated factors change and associations weaken after 65 years of age in both sexes, and that the limited genotypes assessed were secondary to environmental, health, social and behavioral factors.