An 11-gene multiplex polymerase chain reaction (mPCR) was developed based on genes that code for serogroup-specific O-antigens and four major virulence factors (intimin, enterohemorrhagic hemolysin, and Shiga toxins [Stx] 1 and 2), to detect O157 and the "top six" non-O157 (O26, O45, O103, O111, O121, and O145) Shiga toxin-producing Escherichia coli (STEC). The assay specificity was validated with pure cultures of seven major STEC (185 strains), 26 other STEC (65 strains), non-STEC (five strains), and 33 strains of other genera and species. Sensitivity of the assay with cattle fecal sample spiked with pooled cultures of seven major STEC was 10⁵ colony-forming units (CFU)/g before enrichment and 10² CFU/g after enrichment. The applicability of the assay to detect STEC in fecal samples (n=50), before and after enrichment, was evaluated by comparing with culture-based methods for O26, O111, and O157. The mPCR assay of 50 fecal samples showed seven (14%) positive before enrichment and 23 (46%) positive after enrichment for one or more of the seven O-groups. Overall, 17 isolates from 17 fecal samples and 27 isolates (four for O26, three for O45, and 20 for O103) from 19 fecal samples were obtained, by culture-based methods, for O157 and non-O157 serogroups, respectively. None of the 27 non-O157 isolates possessed the stx genes, suggesting that cattle harbor Shiga toxin-negative E. coli belonging to the "top six" non-O157 serogroups. Our data, although based on a limited number of samples, suggest that the sensitivities of the mPCR and culture-based methods in detecting the seven serogroups of STEC in feces differed between O-groups. An obvious limitation of our mPCR is that the concurrent detection of virulence genes and the serogroups in a sample does not necessarily associate the virulence genes with the prevalent serogroups in the same sample. The major application of our 11-gene mPCR assay may be in identifying putative colonies of STEC obtained by culture-based methods.

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The objective of this study was to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) serogroups and associated virulence genes in feces of commercial feedlot cattle. During March to May 2011, fecal samples were collected from individual cattle (n = 960) in 10 cohorts (cattle subpopulations within a feedlot) comprising 17,148 total steers that originated from 48 backgrounding operations in six U.S. states. Fecal samples were enriched in E. coli broth and subjected to two detection protocols: (1) an 11-gene multiplex polymerase chain reaction (PCR) that identifies seven O serogroups (O26, O45, O103, O111, O121, O145, and O157) and four virulence genes (stx1, stx2, eae, and ehxA) applied to extracted total DNA (''direct PCR''); and (2) cultural procedures that involve immunomagnetic separation (IMS) with O26, O103, and O111 beads, plating on a nondifferential MacConkey agar, followed by the multiplex PCR of pooled colonies (''culture-based method''). Generalized linear mixed models were used to adjust prevalence estimates for clustering. Based on direct PCR detection, O157 (49.9%) was the most prevalent O serogroup followed by O26 (20.3%), O103 (11.8%), O121 (10.7%), O45 (10.4%), O145 (2.8%), and O111 (0.8%). Cumulative adjusted prevalence estimates were 22.3, 24.6, and 0.01% for O26, O103, and O111 serogroups, respectively, based on culture-based methods. However, prevalence varied significantly by cohort ( p-values < 0.05) for O26, O121, and O157 based on direct PCR, and for O26, O103, and O111 serogroups based on culture-based methods. Results of this study indicate that all seven STEC serogroups were identified in feedlot cattle feces, with O157, O26, and O103 being the most prevalent serogroups. A substantial proportion of serogroup-positive samples did not harbor Shiga toxin genes; thus, additional elucidation of the potential human health risk is required. Further evaluation of diagnostic methods for non-O157 STEC is needed given their impact on prevalence estimation.

Our primary objective was to determine the efficacy of a siderophore receptor and porin proteins-based vaccine (VAC) and a Lactobacillus acidophilus-based direct-fed microbial (DFM) against fecal shedding of Escherichia coli O157:H7 in commercial feedlot cattle fed a corn grain-based diet with 25% distiller's grains. Cattle projected to be on a finishing diet during the summer were randomly allocated into 40 study pens within ten blocks based on allocation dates. Blocks were complete; each of the four pens within a block was randomly assigned one treatment: control, VAC, DFM, or VAC+DFM. The DFM was fed (10⁶CFU/animal/day of Lactobacillus) throughout the study periods (84-88 days) and cattle were vaccinated at enrollment and again three weeks later. Fresh fecal samples (30/pen) from pen floors were collected weekly for four consecutive weeks (study days 52-77). Two concurrent culture procedures were used to enable estimates of E. coli O157:H7 shedding prevalence and prevalence of high shedders. From 4800 total samples, 1522 (31.7%) were positive for E. coli O157:H7 and 169 (3.5%) were considered high shedders. Pen-level linear mixed models were used for data analyses. There were no significant interactions among treatments and time of sampling. However, vaccinated pens had lower (P<0.01) overall prevalence of E. coli O157:H7 (model-adjusted mean ± SEM=17.4 ± 3.95%) and lower (P<0.01) prevalence of high shedders (0.95 ± 0.26%) than unvaccinated pens (37.0 ± 6.32% and 4.19 ± 0.81%, respectively). There was no evidence of a DFM effect on either measure of E. coli O157:H7 shedding. Results indicate that a two-dose regimen of the vaccine significantly reduces fecal prevalence of E. coli O157:H7 (vaccine efficacy of 53.0%) and prevalence of E. coli O157:H7 high shedders (vaccine efficacy of 77.3%) in commercial feedlot cattle reared in the summer on a finishing diet with 25% distiller's grains.

A multiplex PCR was designed to detect Escherichia coli O104:H4, a hybrid pathotype of Shiga toxigenic and enteroaggregative E. coli, in cattle feces. A total of 248 fecal samples were tested, and 20.6% were positive for serogroup O104. The O104 isolates did not carry genes characteristic of the virulent hybrid strain.