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Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause illnesses in humans ranging from mild to hemorrhagic enteritis with complications of hemolytic uremic syndrome and even death. Cattle are a major reservoir of STEC, which reside in the hindgut and are shed in the feces, a major source of food and water contaminations. Seven serogroups, O26, O45, O103, O111, O121, O145 and O157, called ‘top-7’, are responsible for the majority of human STEC infections in North America. Additionally, 151 serogroups of E. coli are known to carry Shiga toxin genes (stx). Not much is known about fecal shedding and prevalence and virulence potential of STEC other than the top-7. Our primary objectives were to identify serogroups of STEC strains, other than the top-7, isolated from cattle feces and subtype stx genes to assess their virulence potential. Additional objective was to develop and validate a novel multiplex PCR assay to detect and determine prevalence of six serogroups, O2, O74, O109, O131, O168, and O171, in cattle feces. A total of 351 strains, positive for stx gene and negative for the top-7 serogroups, isolated from feedlot cattle feces were used in the study. Of the 351 strains, 291 belonged to 16 serogroups and 60 could not be serogrouped. Among the 351 strains, 63 (17.9%) carried stx1 gene and 300 (82.1%) carried stx2, including 12 strains positive for both. The majority of the stx1 and stx2 were of stx1a (47/63; 74.6%) and stx2a subtypes (234/300; 78%), respectively, which are often associated with human infections. A novel multiplex PCR assay developed and validated to detect six serogroups, O2, O74, O109, O131, O168, and O171, which accounted for 86.9% of the STEC strains identified, was utilized to determine their prevalence in fecal samples (n = 576) collected from a commercial feedlot. Four serogroups, O2, O109, O168, and O171 were identified as the dominant serogroups prevalent in cattle feces. In conclusion, cattle shed in the feces a number of STEC serogroups, other than the top-7, and the majority of the strains isolated possessed stx2, particularly of the subtype 2a, suggesting their potential risk to cause human infections.

Shiga toxin-producing Escherichia coli (STEC) are major foodborne human pathogens that cause mild to hemorrhagic colitis, which could lead to complications of hemolytic uremic syndrome. Seven serogroups, O26, O45, O103, O111, O121, O145, and O157, account for the majority of the STEC illnesses in the United States. Shiga toxins 1 and 2, encoded by stx1 and stx2, respectively, and intimin, encoded by eae gene, are major virulence factors. Cattle are a major reservoir of STEC, but swine also harbor them in the hindgut and shed STEC in the feces. Our objectives were to use a culture method to isolate and identify major and minor serogroups of STEC in finisher pig feces. Shiga toxin genes were subtyped to assess public health implications of STEC. Fecal samples (n = 598) from finisher pigs, collected from 10 pig flows, were enriched in E. coli broth and tested for stx1, stx2, and eae by a multiplex PCR (mPCR) assay. Samples positive for stx1 or stx2 gene were subjected to culture methods, with or without immunomagnetic separation and plating on selective or nonselective media, for isolation and identification of stx-positive isolates. The culture method yielded a total of 178 isolates belonging to 23 serogroups. The three predominant serogroups were O8, O86, and O121. The 178 STEC strains included 26 strains with stx1a and 152 strains with stx2e subtypes. Strains with stx1a, particularly in association with eae (O26 and O103), have the potential to cause severe human infections. All stx2-positive isolates carried the subtype stx2e, a subtype that causes edema disease in swine, but is rarely involved in human infections. Several strains were also positive for genes that encode for enterotoxins, which are involved in neonatal and postweaning diarrhea in swine. In conclusion, our study showed that healthy finisher pigs harbored and shed several serogroups of E. coli carrying virulence genes involved in neonatal diarrhea, postweaning diarrhea, and edema disease, but prevalence of STEC of public health importance was low. HIGHLIGHTS

Escherichia coli carrying prophage with genes that encode for Shiga toxins are categorized as Shiga toxin-producing E. coli (STEC) pathotype. Illnesses caused by STEC in humans, which are often foodborne, range from mild to bloody diarrhea with life-threatening complications of renal failure and hemolytic uremic syndrome and even death, particularly in children. As many as 158 of the total 187 serogroups of E. coli are known to carry Shiga toxin genes, which makes STEC a major pathotype of E. coli. Seven STEC serogroups, called top-7, which include O26, O45, O103, O111, O121, O145, and O157, are responsible for the majority of the STEC-associated human illnesses. The STEC serogroups, other than the top-7, called “non-top-7” have also been associated with human illnesses, more often as sporadic infections. Ruminants, particularly cattle, are principal reservoirs of STEC and harbor the organisms in the hindgut and shed in the feces, which serves as a major source of food and water contaminations. A number of studies have reported on the fecal prevalence of top-7 STEC in cattle feces. However, there is paucity of data on the prevalence of non-top-7 STEC serogroups in cattle feces, generally because of lack of validated detection methods. The objective of our study was to develop and validate 14 sets of multiplex PCR (mPCR) assays targeting serogroup-specific genes to detect 137 non-top-7 STEC serogroups previously reported to be present in cattle feces. Each assay included 7–12 serogroups and primers were designed to amplify the target genes with distinct amplicon sizes for each serogroup that can be readily identified within each assay. The assays were validated with 460 strains of known serogroups. The multiplex PCR assays designed in our study can be readily adapted by most laboratories for rapid identification of strains belonging to the non-top-7 STEC serogroups associated with cattle.

Campylobacter spp. can be pathogenic to humans and often harbor antimicrobial resistance genes. Data on resistance in relation to fluoroquinolone use in beef cattle are scarce. This cross-sectional study of preharvest cattle evaluated Campylobacter prevalence and susceptibility to nalidixic acid and ciprofloxacin in feedlots that previously administered a fluoroquinolone as primary treatment for bovine respiratory disease. Twenty fresh fecal samples were collected from each of 10 pens, in each of five feedlots, 1-2 weeks before harvest. Feces were cultured for Campylobacter using selective enrichment and isolation methods. Genus and species were confirmed via PCR. Minimum inhibitory concentrations (MICs) of ciprofloxacin and nalidixic acid were determined using a micro-broth dilution method and human breakpoints. Antimicrobial use within each pen was recorded. Data were analyzed using generalized linear mixed-models (prevalence) and survival analysis (MICs). Overall, sample-level prevalence of Campylobacter was 27.2% (272/1000) and differed significantly among feedlots (p 0.01). Campylobacter coli was the most common species (55.1%; 150/272), followed by Campylobacter hyointestinalis (42.6%; 116/272). Within-pen prevalence was not significantly associated with the number of fluoroquinolone treatments, sex, body weight, or metaphylaxis use, but was associated with the number of days cattle were in the feedlot (p = 0.03). The MICs for the majority of Campylobacter isolates were above the breakpoints for nalidixic acid (68.4%; 175/256) and for ciprofloxacin (65.6%; 168/256). Distributions of MICs for nalidixic acid (p ≤ 0.01) and ciprofloxacin (p ≤ 0.05) were significantly different among feedlots, and by Campylobacter species. However, fluoroquinolone treatments, sex, body weight, days on feed, and metaphylaxis were not significantly associated with MIC distributions within pens. We found no evidence that the number of fluoroquinolone treatments within feedlot pens significantly affected the within-pen fecal prevalence or quinolone susceptibilies of Campylobacter in feedlots that used a fluoroquinolone as primary treatment for bovine respiratory disease.

Shiga toxin-producing E. coli (STEC) are major foodborne pathogens. While many studies have focused on the "top-7 STEC", little is known for minor serogroups. A total of 284 non-top-7 STEC strains isolated from cattle feces were subjected to whole-genome sequencing (WGS) to determine the serotypes, the presence of virulence genes and antimicrobial resistance (AMR) determinants. Nineteen typeable and three non-typeable serotypes with novel O-antigen loci were identified. Twenty-one AMR genes and point mutations in another six genes that conferred resistance to 10 antimicrobial classes were detected, as well as 46 virulence genes. The distribution of 33 virulence genes and 15 AMR determinants exhibited significant differences among serotypes (p 0.05). Among all strains, 81.7% (n = 232) and 14.1% (n = 40) carried stx2 and stx1 only, respectively; only 4.2% (n = 12) carried both. Subtypes stx1a, stx1c, stx2a, stx2c, stx2d, and stx2g were identified. Forty-six strains carried eae and stx2a and therefore had the potential cause severe diseases; 47 strains were genetically related to human clinical strains inferred from a pan-genome phylogenetic tree. We were able to demonstrate the utility of WGS as a surveillance tool to characterize the novel serotypes, as well as AMR and virulence profiles of uncommon STEC that could potentially cause human illness.

Escherichia coli serogroups O157, O26, O45, O103, O111, O121, and O145, when carrying major virulence genes, the Shiga toxin genes stx(1) and stx(2) and the intimin gene eae, are important foodborne pathogens. They are referred to as the “top 7” Shiga toxin-producing E. coli (STEC) serogroups and were declared by the USDA as adulterants to human health. Since top 7 serogroup-positive cattle feces and ground beef can also contain nonadulterant E. coli strains, regular PCR cannot confirm whether the virulence genes are carried by adulterant or nonadulterant E. coli serogroups. Thus, traditional gold-standard STEC detection requires bacterial isolation and characterization, which are not compatible with high-throughput settings and often take a week to obtain a definitive result. In this study, we demonstrated that the partition-based multichannel digital PCR (dPCR) system can be used to detect and associate the E. coli serogroup-specific gene with major virulence genes and developed a single-cell-based dPCR approach for rapid (within 1 day) and accurate detection and confirmation of major STEC serogroups in high-throughput settings. Major virulence genes carried by each of the top 7 STEC serogroups were detected by dPCR with appropriately diluted intact bacterial cells from pure cultures, culture-spiked cattle feces, and culture-spiked ground beef. Furthermore, from 100 randomly collected, naturally shed cattle fecal samples, 3 O103 strains carrying eae and 2 O45 strains carrying stx(1) were identified by this dPCR assay and verified by the traditional isolation method. This novel and rapid dPCR assay is a culture-independent, high-throughput, accurate, and sensitive method for STEC detection and confirmation.

Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, referred to as the top six non-O157 serogroups, are responsible for more than 70% of human non-O157 STEC infections in North America. Cattle harbor non-O157 strains in the hindgut and shed them in the feces. The objective of this study was to use the U.S. Food and Drug Administration (FDA) E. coli identification (ECID) DNA microarray to identify the serotype, assess the virulence potential of each, and determine the phylogenetic relationships among five of the six non-O157 E. coli serogroups isolated from feedlot cattle feces. Forty-four strains of STEC, enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), or putative nonpathotype E. coli (NPEC) of cattle origin and five human clinical strains of EHEC were assayed with the FDA-ECID DNA microarray. The cattle strains harbored diverse flagellar genes. The bovine and human strains belonging to serogroups O26, O45, and O103 carried stx

Escherichia coli O145 serogroup is one of the big six non-O157 Shiga toxin producing E. coli (STEC) that causes foodborne illnesses in the United States and other countries. Cattle are a major reservoir of STEC, which harbor them in their hindgut and shed in the feces. Cattle feces is the main source of hide and subsequent carcass contaminations during harvest leading to foodborne illnesses in humans. The objective of our study was to determine the virulence potential of STEC O145 strains isolated from cattle feces and hide samples. A total of 71 STEC O145 strains isolated from cattle feces (n = 16), hide (n = 53), and human clinical samples (n = 2) were used in the study. The strains were subjected to whole genome sequencing using Illumina MiSeq platform. The average draft genome size of the fecal, hide, and human clinical strains were 5.41, 5.28, and 5.29 Mb, respectively. The average number of genes associated with mobile genetic elements was 260, 238, and 259, in cattle fecal, hide, and human clinical strains, respectively. All strains belonged to O145:H28 serotype and carried eae subtype γ. Shiga toxin 1a was the most common Shiga toxin gene subtype among the strains, followed by stx2a and stx2c. The strains also carried genes encoding type III secretory system proteins, nle, and plasmid-encoded virulence genes. Phylogenetic analysis revealed clustering of cattle fecal strains separately from hide strains, and the human clinical strains were more closely related to the hide strains. All the strains belonged to sequence type (ST)-32. The virulence gene profile of STEC O145 strains isolated from cattle sources was similar to that of human clinical strains and were phylogenetically closely related to human clinical strains. The genetic analysis suggests the potential of cattle STEC O145 strains to cause human illnesses. Inclusion of more strains from cattle and their environment in the analysis will help in further elucidation of the genetic diversity and virulence potential of cattle O145 strains.

Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, called non-O157 STEC, are important foodborne pathogens. Cattle, a major reservoir, harbor the organisms in the hindgut and shed them in the feces. Although limited data exist on fecal shedding, concentrations of non-O157 STEC in feces have not been reported. The objectives of our study were (i) to develop and validate two multiplex quantitative PCR (mqPCR) assays, targeting O-antigen genes of O26, O103, and O111 (mqPCR-1) and O45, O121, and O145 (mqPCR-2); (ii) to utilize the two assays, together with a previously developed four-plex qPCR assay (mqPCR-3) targeting the O157 antigen and three virulence genes (stx1, stx2, and eae), to quantify seven serogroups and three virulence genes in cattle feces; and (iii) to compare the three mqPCR assays to a 10-plex conventional PCR (cPCR) targeting seven serogroups and three virulence genes and culture methods to detect seven E. coli serogroups in cattle feces. The two mqPCR assays (1 and 2) were shown to be specific to the target genes, and the detection limits were 4 and 2 log CFU/g of pure culture-spiked fecal samples, before and after enrichment, respectively. A total of 576 fecal samples collected from a feedlot were enriched in E. coli broth and were subjected to quantification (before enrichment) and detection (after enrichment). Of the 576 fecal samples subjected, before enrichment, to three mqPCR assays for quantification, 175 (30.4%) were quantifiable (≥4 log CFU/g) for at least one of the seven serogroups, with O157 being the most common serogroup. The three mqPCR assays detected higher proportions of postenriched fecal samples (P0.01) as positive for one or more serogroups compared with cPCR and culture methods. This is the first study to assess the applicability of qPCR assays to detect and quantify six non-O157 serogroups in cattle feces and to generate data on fecal concentration of the six serogroups.

Shiga toxin-producing Escherichia coli (STEC) of the serogroups O26, O45, O103, O111, O121, and O145, often called non-O157 STEC, are foodborne pathogens. Cattle are asymptomatic reservoirs for STEC; the organisms reside in the hindgut and are shed in the feces, which serve as the source of food product contaminations. Culture-based detection of non-O157 STEC involves an immunomagnetic separation (IMS) step to capture the specific serogroups in complex matrices, such as feces. The IMS procedure is time consuming and labor intensive because of the need to subject each fecal sample to six individual beads. Therefore, our objective was to evaluate whether pooling of IMS beads affects sensitivity of non-O157 STEC detection compared with using individual IMS beads. The evaluation was done by comparing detection of serogroups in feces spiked with pure cultures (experiments 1 and 2) and from feces (n = 384) of naturally shedding cattle (experiment 3). In spiked fecal samples, detection with pools of three, four, six, or seven beads was similar to, or at times higher than, detection with individual IMS beads. In experiment 3, the proportions of fecal samples that tested positive for the six serogroups as detected by individual or pooled beads were similar. Based on noninferiority tests, detection with pooled beads was not substantially inferior to detection with individual beads (P0.05). In conclusion, the pooling of IMS beads is a better option for detection of STEC serogroups in fecal samples compared with individual beads because the procedure saves time and labor and has the prospect of a higher throughput.

Probiotics, an antibiotic alternative, are widely used as feed additives for performance benefits in cattle and swine production systems. Among bacterial species contained in probiotics, Enterococcus faecium is common. Antimicrobial resistance (AMR), particularly multidrug resistance, is a common trait among enterococci because of their propensity to acquire resistance and horizontally transfer AMR genes. Also, E. faecium is an opportunistic pathogen, and in the United States, it is the second most common nosocomial pathogen. There has been no published study on AMR and virulence potential in E. faecium contained in probiotic products used in cattle and swine in the United States. Therefore, our objectives were to determine phenotypic susceptibilities or resistance to antimicrobials, virulence genes (asa1, gelE, cylA, esp, and hyl) and assess genetic diversity of E. faecium isolated from commercial products. Twenty-two commercially available E. faecium-based probiotic products used in cattle (n = 13) and swine (n = 9) were procured and E. faecium was isolated and species confirmed. Antimicrobial susceptibility testing to determine minimum inhibitory concentrations was done by micro-broth dilution method using National Antimicrobial Resistance Monitoring Systems Gram-positive Sensititre panel plate (CMV3AGPF), and categorization of strains as susceptible or resistant was as per Clinical Laboratory and Standards Institute's guidelines. E. faecium strains from 7 products (3 for swine and 4 for cattle) were pan-susceptible to the 16 antimicrobials tested. Strains from 15 products (6 for swine and 9 for cattle) exhibited resistance to at least one antimicrobial and a high proportion of strains was resistant to lincomycin (10/22), followed by tetracycline (4/22), daptomycin (4/22), ciprofloxacin (4/22), kanamycin (3/22), and penicillin (2/22). Four strains were multidrug resistant, with resistant phenotypes ranging from 3 to 6 antimicrobials or class. None of the E. faecium strains were positive for any of the virulence genes tested. The clonal relationships among the 22 E. faecium strains were determined by pulsed-field gel electrophoresis (PFGE) typing. A total of 10 PFGE patterns were observed with 22 strains and a few of the strains from different probiotic products had identical (100% Dice similarity) PFGE patterns. In conclusion, the E. faecium strains in a few commercial probiotics exhibited AMR to medically-important antimicrobials, but none contained virulence genes.

Escherichia coli O103, harbored in the hindgut and shed in the feces of cattle, can be enterohemorrhagic (EHEC), enteropathogenic (EPEC), or putative non-pathotype. The genetic diversity particularly that of virulence gene profiles within O103 serogroup is likely to be broad, considering the wide range in severity of illness. However, virulence descriptions of the E. coli O103 strains isolated from cattle feces have been primarily limited to major genes, such as Shiga toxin and intimin genes. Less is known about the frequency at which other virulence genes exist or about genes associated with the mobile genetic elements of E. coli O103 pathotypes. Our objective was to utilize whole genome sequencing (WGS) to identify and compare major and putative virulence genes of EHEC O103 (positive for Shiga toxin gene, stx1, and intimin gene, eae; n = 43), EPEC O103 (negative for stx1 and positive for eae; n = 13) and putative non-pathotype O103 strains (negative for stx and eae; n = 13) isolated from cattle feces. Six strains of EHEC O103 from human clinical cases were also included. All bovine EHEC strains (43/43) and a majority of EPEC (12/13) and putative non-pathotype strains (12/13) were O103:H2 serotype. Both bovine and human EHEC strains had significantly larger average genome sizes (P < 0.0001) and were positive for a higher number of adherence and toxin-based virulence genes and genes on mobile elements (prophages, transposable elements, and plasmids) than EPEC or putative non-pathotype strains. The genome size of the three pathotypes positively correlated (R2 = 0.7) with the number of genes carried on mobile genetic elements. Bovine strains clustered phylogenetically by pathotypes, which differed in several key virulence genes. The diversity of E. coli O103 pathotypes shed in cattle feces is likely reflective of the acquisition or loss of virulence genes carried on mobile genetic elements.

The study objective was to determine effects of fluoroquinolone metaphylaxis on fecal prevalence of Salmonella and Campylobacter and fecal prevalence of quinolone-resistant Salmonella and Campylobacter in feedlot cattle. On Day 0, cattle (n = 288) at risk for bovine respiratory disease (BRD) were randomly assigned to either a nontreated control pen (12 pens) or a fluoroquinolone-treated (enrofloxacin; Baytril® 100) pen (12 pens). Rectal fecal samples were collected from cattle on days 0, 7, 14, 21, and 28. Feces were cultured for Salmonella enterica and Campylobacter spp. using enrichment and selective isolation methods, and confirmed by serology and PCR. Susceptibilities to nalidixic acid and ciprofloxacin were determined using microbroth dilution methods. Data analyses were performed using linear mixed models. Overall, Salmonella sp. and Campylobacter spp. were recovered from 10.2% (139/1,364) and 12.4% (170/1,364) of the fecal samples, respectively. Campylobacter species included hyointestinal...

Shiga toxin producing Escherichia coli (STEC) are important foodborne pathogens responsible for human illnesses. Cattle are a major reservoir that harbor the organism in the hindgut and shed in the feces. Shiga toxins (Stx) are the primary virulence factors associated with STEC illnesses. The two antigenically distinct Stx types, Stx1 and Stx2, encoded by stx1 and stx2 genes, share approximately 56% amino acid sequence identity. Genetic variants exist within Stx1 and Stx2 based on differences in amino acid composition and in cytotoxicity. The objective of our study was to identify the stx subtypes in strains of STEC serogroups, other than O157, isolated from cattle feces. Shiga toxin gene carrying E. coli strains (n = 192), spanning 27 serogroups originating from cattle (n = 170) and human (n = 22) sources, were utilized in the study. Shiga toxin genes were amplified by PCR, sequenced, and nucleotide sequences were translated into amino acid sequences using CLC main workbench software. Shiga toxin subtypes were identified based on the amino acid motifs that define each subtype. Shiga toxin genotypes were also identified at the nucleotide level by in silico restriction fragment length polymorphism (RFLP). Of the total 192 STEC strains, 93 (48.4%) were positive for stx1 only, 43 (22.4%) for stx2 only, and 56 (29.2%) for both stx1 and stx2. Among the 149 strains positive for stx1, 132 (88.6%) were stx1a and 17 (11.4%) were stx1c. Shiga toxin 1a was the most common subtype of stx1 among cattle (87.9%; 123/140) and human strains (100%; 9/9) of non-O157 serogroups. Of the total 99 strains positive for stx2, 79 were stx2a (79.8%), 11 (11.1%) were stx2c, 12 (12.1%) were stx2d. Of the 170 strains originating from cattle feces, 58 (34.1%) were stx2a subtype, 11 (6.5%) were stx2c subtype, and 11 were of subtype stx2d (6.5%). All but one of the human strains were positive for stx2a. Three strains of cattle origin were positive for both stx2a and stx2d. In conclusion, a number of non-O157 STEC serogroups harbored by cattle possess a wide variety of Shiga toxin subtypes, with stx1a and stx2a being the most predominant stx subtypes occurring individually or in combination. Cattle are a reservoir of a number of non-O157 STEC serogroups and information on the Shiga toxin subtypes is useful in assessing the potential risk as human pathogens.

Escherichia coli O26 is second only to O157 in causing foodborne, Shiga toxin-producing E. coli (STEC) infections. Our objectives were to determine fecal prevalence and characteristics of E. coli O26 in commercial feedlot cattle (17,148) that were enrolled in a study to evaluate an E. coli O157:H7 siderophore receptor and porin (SRP(®)) vaccine (VAC) and a direct-fed microbial (DFM; 10(6) colony-forming units [CFU]/animal/day of Lactobacillus acidophilus and 10(9) CFU/animal/day of Propionibacterium freudenreichii). Cattle were randomly allocated to 40 pens within 10 complete blocks; pens were randomly assigned to control, VAC, DFM, or VAC+DFM treatments. Vaccine was administered on days 0 and 21, and DFM was fed throughout the study. Pen-floor fecal samples (30/pen) were collected weekly for the last 4 study weeks. Samples were enriched in E. coli broth and subjected to a multiplex polymerase chain reaction (PCR) designed to detect O26-specific wzx gene and four major virulence genes (stx1, stx2, eae, and ehxA) and to a culture-based procedure that involved immunomagnetic separation and plating on MacConkey agar. Ten presumptive E. coli colonies were randomly picked, pooled, and tested by the multiplex PCR. Pooled colonies positive for O26 serogroup were streaked on sorbose MacConkey agar, and 10 randomly picked colonies per sample were tested individually by the multiplex PCR. The overall prevalence of E. coli O26 was higher (p0.001) by the culture-based method compared to the PCR assay (22.7 versus 10.5%). The interventions (VAC and or DFM) had no impact on fecal shedding of O26. Serogroup O26 was recovered in pure culture from 23.9% (260 of 1089) of O26 PCR-positive pooled colonies. Only 7 of the 260 isolates were positive for the stx gene and 90.1% of the isolates possessed an eaeβ gene that codes for intimin subtype β, but not the bfpA gene, which codes for bundle-forming pilus. Therefore, the majority of the O26 recovered from feedlot cattle feces was atypical enteropathogenic E. coli, and not STEC.

Shiga toxin-producing Escherichia coli (STEC) are important human pathogens, and attention to non-O157 serogroups has increased in recent years. Although cattle are normally considered the primary reservoir for STEC, recent illnesses associated with goat contact have indicated that these animals are important potential reservoirs for the organisms. The prevalence of STEC, particularly non-O157 serogroups, in U.S. goats has not been well described. Our objective was to determine the prevalence of six major non-O157 STEC serogroups in the feces of meat goats. Rectal contents from 296 goats were collected postevisceration at a slaughter plant in the southeastern United States over 9 days during a 12-week period from August through October 2012. Samples were enriched in E. coli broth, and DNA was extracted and used as template in an 11-gene multiplex PCR that detected six non-O157 serogroups (O26, O45, O103, O121, O111, and O145) and virulence genes. Samples were considered positive when at least one non-O157 STEC serotype was present with either stx 1 or stx 2. All six non-O157 serogroups were detected by PCR in our samples, and 14.5% of samples were positive for at least one serogroup. Prevalence of O26 was highest, with 6.4% of goat fecal samples positive. The prevalence of O45 was 3.4%, O103 was 4.4%, O111 was 4.1%, O121 was 1.4%, and O145 was 3.0%. Twenty-two (7.4%) of 296 fecal samples had more than one non-O157 serogroup detected in the feces. Two samples had evidence of three non-O157 STEC serogroups. Goats appear to be an important reservoir for non-O157 STEC, and further work to understand the characteristics, epidemiology, and ecology of STEC in these animals is warranted.

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.

Salmonella is an important foodborne pathogen and antimicrobial resistance can be a human health concern. The objectives of this cross-sectional study were to (1) determine the prevalence and quinolone susceptibility of Salmonella in feces of preharvest commercial feedlot cattle and (2) determine if the prevalence and susceptibility of Salmonella isolates were associated with previous fluoroquinolone use within pens. Five feedlots in western Kansas and Texas were selected based on their use of a commercially licensed fluoroquinolone for initial treatment of bovine respiratory disease (BRD). Twenty pen floor fecal samples were collected from each of 10 pens from each feedlot during early summer of 2012. Salmonella isolation was performed and microbroth dilution was used to determine susceptibility of isolates to nalidixic acid and ciprofloxacin. Prior antimicrobial treatment data were retrieved from feedlots' operational data. Generalized linear mixed models were used to assess associations between Salmonella prevalence and the number of fluoroquinolone treatments within pens while taking into consideration cattle demographic and management factors, as well as the hierarchical structure of the data. Overall, cumulative fecal prevalence of Salmonella was 38.0% (380/1000), but prevalence varied significantly (p

Cattle are asymptomatic reservoirs for Escherichia coli O157, a major foodborne pathogen. The organism generally colonizes the hindgut of cattle and is shed in the feces at low concentrations. The objective of this research was to evaluate a multiplex, real-time polymerase chain reaction (mqPCR) assay for quantification of E. coli O157 in cattle feces using stx1, stx2, and rfbE gene targets. Primer efficiency and analytical sensitivity of the assay were evaluated with a single or pooled (five strain) culture of E. coli O157. In pure culture, the minimum detection limit of the assay was 1.4×10(3) CFU/mL and 3.6×10(3) CFU/mL for the single and five-strain mixture of E. coli O157, respectively. Diagnostic sensitivity was analyzed using DNA extracted from cattle feces spiked with E. coli O157. In feces spiked with the pooled mixture of five E. coli O157 strains, the minimum detection limit was 3.6×10(4) CFU/g. We also evaluated the assay with feces from cattle experimentally inoculated with E. coli O157 by comparing the results to a culture-based method. For the majority of samples tested, the concentration of E. coli O157 detected by the real-time and culture methods was within one log difference. However, the assay could only be evaluated for cattle shedding high concentrations of E. coli O157. In conclusion, the mqPCR quantifying E. coli O157 in cattle feces using stx1, stx2, and rfbE gene targets may have use in detecting and quantifying super shedders, but is not applicable for quantification in animals shedding low concentrations (10(2) to 10(3) CFU/g feces).

Our objectives were to determine factors associated with fecal prevalence of Salmonella at feedlot entry and within 24 h of harvest (preharvest), and to assess potential persistence of Salmonella strains within cattle populations. This repeated cross-sectional study followed 5559 beef cattle within 30 feedlot cohorts. Samples (n = 30) of fresh feces were collected from the pen floor of each cohort at feedlot entry and preharvest. Samples were subjected to a selective Salmonella isolation protocol and serotypes were determined for Salmonella isolates. Genetic similarity of a subset of isolates was determined using pulsed-field gel electrophoresis (PFGE). Cattle health and performance data were recorded electronically by feedlot personnel. Cohort-level generalized linear mixed models were used to assess bivariable associations. Fecal prevalence of Salmonella within a cohort at feedlot entry (mean = 64.7%) was not associated with preharvest prevalence (mean = 72.6%). Prevalence at feedlot entry was negatively associated with mean entry weight (p = 0.02). Preharvest prevalence was positively associated with the number of days in the feedlot (p = 0.02), cumulative morbidity (p = 0.01), and cumulative mortality (p = 0.03). We recovered Salmonella isolates with identical PFGE profiles both at feedlot entry and preharvest from 14 cohorts of cattle. Fecal prevalence of Salmonella immediately before harvest may be higher in subsets of the feedlot population, but does not appear to be affected by prevalence at feedlot entry. However, PFGE subtypes of Salmonella appear to persist within and among feedlot cohorts throughout the feeding period.

Escherichia coli serotype O157:H7 continues to pose a serious health threat to human beings. Cattle, a major reservoir of the pathogen, harbor E. coli O157:H7 in their gastrointestinal tract and shed variable concentrations of E. coli O157:H7 into the environment. Genetic characterization of cattle-shed E. coli O157 strains is of interest to the livestock industry, food business, and public health community. The present study applied whole genome shotgun sequencing (WGS) and single nucleotide variant (SNV) calling to characterize 279 cattle-shed E. coli O157:H7 strains isolated from a single feedlot located in southwestern region of the US. More than 4,000 SNVs were identified among the strains and the resultant phylogenomic tree revealed three major groups. Using the Sakai strain genome as reference, more than 2,000 SNVs were annotated and a detailed SNV map generated. Results clearly revealed highly polymorphic loci along the E. coli O157:H7 genome that aligned with the prophage regions and highly variant genes involved in processing bacterial genetic information. The WGS data were further profiled against a comprehensive virulence factor database (VFDB) for virulence gene identification. Among the total 285 virulence genes identified, only 132 were present in all the strains. There were six virulence genes unique to single isolates. Our findings suggested that the genome variations of the E. coli O157:H7 were mainly attributable to dynamics of certain phages, and the bacterial strains have variable virulence gene profiles, even though they came from a single cattle population, which may explain the differences in pathogenicity, host prevalence, and transmissibility by E. coli O157:H7.

A multiplex PCR procedure that detects six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in Escherichia coli O157:H7 was developed. Analyses of the available sequences of the six major virulence genes and the published primers allowed us to develop the six-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting six bands for fliC, stx1, stx2, eae, rfbE, and hlyA were even and distinct with product sizes of 949, 655, 477, 375, 296, and 199 bp, respectively. The procedure was validated with a total of 221 E. coli strains that included 4 ATCC, 84 cattle, and 57 human E. coli O157:H7 strains as well as 76 non-O157 cattle and human E. coli strains. The results of all 221 strains were similar to the results generated by established multiplex PCR methods that involved two separate reactions to detect five virulence genes (stx1, stx2, eae, fliC, and hlyA). Specificity of the O antigen was indicated by amplification of only O157, and not O25, O26, O55, O78, O103, O111, O127, and O145 E. coli serotypes. Sensitivity tests showed that the procedure amplified genes from a fecal sample spiked with a minimum of 104 CFU/g (10 cells/reaction) of E. coli O157. After a 6-h enrichment of E. coli O157-spiked samples, a sensitivity level of 10 CFU/g was achieved.

Escherichia coli O104:H4, a hybrid pathotype reported in a large 2011 foodborne outbreak in Germany, has not been detected in cattle feces. However, cattle harbor and shed in the feces other O104 serotypes, particularly O104:H7, which has been associated with sporadic cases of diarrhea in humans. The objective of our study was to assess the virulence potential of Shiga toxin-producing E. coli (STEC) O104:H7 isolated from feces of feedlot cattle using DNA microarray. Six strains of STEC O104:H7 isolated from cattle feces were analyzed using FDA-E. coli Identification (ECID) DNA microarray to determine their virulence profiles and compare them to the human strains (clinical) of O104:H7, STEC O104:H4 (German outbreak strain), and O104:H21 (milk-associated Montana outbreak strain). Scatter plots were generated from the array data to visualize the gene-level differences between bovine and human O104 strains, and Pearson correlation coefficients (r) were determined. Splits tree was generated to analyze relatedness between the strains. All O104:H7 strains, both bovine and human, similar to O104:H4 and O104:H21 outbreak strains were negative for intimin (eae). The bovine strains were positive for Shiga toxin 1 subtype c (stx1c), enterohemolysin (ehxA), tellurite resistance gene (terD), IrgA homolog protein (iha), type 1 fimbriae (fimH), and negative for genes that code for effector proteins of type III secretory system. The six cattle O104 strains were closely related (r = 0.86-0.98) to each other, except for a few differences in phage related and non-annotated genes. One of the human clinical O104:H7 strains (2011C-3665) was more closely related to the bovine O104:H7 strains (r = 0.81-0.85) than the other four human clinical O104:H7 strains (r = 0.75-0.79). Montana outbreak strain (O104:H21) was more closely related to four of the human clinical O104:H7 strains than the bovine O104:H7 strains. None of the bovine E. coli O104 strains carried genes characteristic of E. coli O104:H4 German outbreak strain and unlike other human strains were also negative for Shiga toxin 2. Because cattle E. coli O104:H7 strains possess stx1c and genes that code for enterohemolysin and a variety of adhesins, the serotype has the potential to be a diarrheagenic foodborne pathogen in humans.

Our objective was to define and compare pulsed-field gel electrophoresis (PFGE) profiles of Escherichia coli O157 isolated from cattle feces and carcass samples to evaluate relationships between beef carcass contamination and fecal shedding of E. coli O157 at harvest. We used PFGE separation of Xba1-digested DNA to characterize E. coli O157 isolates (n = 174) from preevisceration carcasses (n = 39) and feces (n = 135) that were recovered from 37 E. coli O157-positive truckloads sampled at a commercial abattoir. Semiquantitative fecal culture techniques differentiated high-shedding, low-shedding, and negative cattle. Among all isolates, there were 17 PFGE types (95% homology) and 37 subtypes (100% homology). Specific subtypes were detected on multiple occasions and from different sample types within loads, among loads, and among days. Seventeen subtypes were recovered from carcasses; most were also recovered from feces of high-shedding cattle (13) and low-shedding cattle (14). Within truckload, the percentages of carcass isolates that were identical to high-shedder or low-shedder fecal isolates, as determined by PFGE, were 69.2% and 46.0%, respectively, whereas among different truckloads within the same study day, the percentages of carcass isolates that were the same subtype as high-shedder or low-shedder fecal isolates were 35.3% and 58.8%, respectively. Our results suggest that cattle feces from both low- and high-shedders pose a potential risk for E. coli O157 contamination of carcasses. Truckload may be an important factor in the potential transmission of E. coli O157, but isolates from carcasses also may be similar to those from feces of cattle on different truckloads and harvest days.

Fecal shedding of Escherichia coli O157:H7 in cattle, except those that shed transiently, is due to the organism's ability to persist in the gut. Site of prevalence in the gut is important for understanding the mechanisms and factors affecting gut persistence and fecal shedding and is a potential target for intervention. The prevalence of E. coli O157:H7 in the rumen, cecum, colon, and rectum was determined with contents collected from slaughtered cattle (n = 815) at an abattoir. Isolation and identification of E. coli O157:H7 were by selective enrichment, immunomagnetic separation, plating on selective medium, agglutination for O157 antigen, and presence of virulence genes. Prevalence in the rumen, cecum, colon, and rectum was 4.9%, 9.9%, 7.6%, and 11.1%, respectively. The overall prevalence of E. coli O157:H7 in the cattle sampled, based on being positive in any one gut location, was 20.3%. E. coli O157:H7 in rectal contents was positively associated (p < 0.01) with presence in the rumen or colon but not in the cecum. Pulsed-field gel electrophoresis (PFGE) was performed to compare the clonal similarity of isolates (n = 144) obtained from the rectum with that of rumen, cecum, or colon within cattle (n = 77). The majority (79-90%) of isolates obtained within the same animal shared a common PFGE type. There were no significant differences in PFGE type between positive samples from the rectum and samples from other locations within the same animal. Acid tolerance for cattle with positive rumen (pregastric) isolates and with at least one other positive hindgut (postgastric) isolate within the same animal was determined. There was no significant difference between gut locations in log reduction following acid challenge. The hindgut was the major site of prevalence of E. coli O157:H7 in cattle, a majority of the isolates within the same animal were clonally similar, and acid tolerance of hindgut isolates were not different from that of ruminal isolates.

Several real-time polymerase chain reaction (PCR) assays have been developed to detect and quantify Shiga toxin-producing Escherichia coli (STEC) O157:H7, but none have targeted the O-antigen specific gene (rfbEO157) in combination with the three major virulence genes, stx1, stx2, and eae. Our objectives were to develop and validate a four-plex, quantitative PCR (mqPCR) assay targeting rfbE(O157), stx1, stx2, and eae for the detection and quantification of STEC O157 in cattle feces, and compare the applicability of the assay to detect STEC O157 to a culture method and conventional PCR (cPCR) targeting the same four genes. Specificity of the mqPCR assay to differentially detect the four genes was confirmed with strains of O157 and non-O157 STEC with different profiles of target genes. In cattle feces spiked with pure cultures, detection limits were 2.8×10(4) and 2.8×10(0) colony-forming units/g before and after enrichment, respectively. Detection of STEC O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. The mqPCR detected 48.9% (136/278) of samples as positive for E. coli O157. Of the 100 samples that were randomly picked from 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar's chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect STEC O157 in cattle feces. However, the use of real-time PCR as a screening method to identify positive samples and then subjecting only positive samples to a culture method may underestimate the presence of STEC O157 in fecal samples.

This study investigated the prevalence and distribution of Escherichia coli O157 genetic types within and among feedlots using pulsed-field gel electrophoresis to separate XbaI-digested DNA. The study population consisted of 300 pens of cattle in 30 feedlots in Kansas that were sampled (feces, water, and water sediment) within a month of being shipped for slaughter. The prevalence of E. coli O157 was 8.5% in feces, 3.1% in water, and 4.5% in water sediment samples. A total of 424 E. coli O157 isolates were characterized by pulsed-field gel electrophoresis, and 139 subtypes (100% Dice similarity with no band differences) were identified. The majority of subtypes (70/139) was identified only once, but nine were identified 10 or more times. Identical subtypes were recovered from both feces and water tanks in 10 feedlots. The majority of subtypes were identified in only one feedlot, and the number of subtypes ranged from one to 23 within a feedlot and from one to seven within a pen. There were 10 feedlots with at least 15 positive samples. In these 10 feedlots, the most common subtype accounted for 16.9-78.6% of the isolates. Common subtypes differed among feedlots. In eight of the 10 feedlots, the most common subtype was identified in multiple pens. The results support a complex ecology for E. coli O157 in feedlot operations, with factors associated with exposure and transmission likely acting at a common level for multiple feedlots, within feedlots, and within pens of cattle.

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 (

Detection of Escherichia coli O157 in cattle feces has traditionally used culture-based methods; PCR-based methods have been suggested as an alternative. We aimed to determine if multiplex real-time (mq) or conventional PCR methods could reliably detect cattle naturally shedding high (≥10(4) CFU/g of feces) and low (∼10(2) CFU/g of feces) concentrations of E. coli O157. Feces were collected from pens of feedlot cattle and evaluated for E. coli O157 by culture methods. Samples were categorized as (i) high shedders, (ii) immunomagnetic separation (IMS) positive after enrichment, or (iii) culture negative. DNA was extracted pre- and postenrichment from 100 fecal samples from each category (high shedder, IMS positive, culture negative) and subjected to mqPCR and conventional PCR assays based on detecting three genes, rfbE, stx1, and stx2. In feces from cattle determined to be E. coli O157 high shedders by culture, 37% were positive by mqPCR prior to enrichment; 85% of samples were positive after enrichment. In IMS-positive samples, 4% were positive by mqPCR prior to enrichment, while 43% were positive after enrichment. In culture-negative feces, 7% were positive by mqPCR prior to enrichment, and 40% were positive after enrichment. The proportion of high shedder-positive and culture-positive (high shedder and IMS) samples were significantly different from mqPCR-positive samples before and after enrichment (P0.01). Similar results were observed for conventional PCR. Our data suggest that mqPCR and conventional PCR are most useful in identifying high shedder animals and may not be an appropriate substitute to culture-based methods for detection of E. coli O157 in cattle feces.

Gallbladders and rectal contents were collected from cattle ( n = 933) at slaughter to determine whether the gallbladder harbors Escherichia coli O157:H7. Both gallbladder mucosal swabs and homogenized mucosal tissues were used for isolation. Only five gallbladders (0.54%) were positive for E. coli O157:H7. Fecal prevalence averaged 7.1%; however, none of the cattle that had E. coli O157:H7 in the gallbladder was positive for E. coli O157:H7 in feces. Therefore, the gallbladder does not appear to be a common site of colonization for E. coli O157:H7 in beef cattle.

We compared fecal samples with samples collected with rectoanal mucosa swabs (RAMS) to determine the prevalence of Escherichia coli O157 in feedlot cattle ( n = 747). Escherichia coli O157 was detected in 9.5% of samples collected with RAMS and 4.7% of samples tested by fecal culture. Pulsed-field gel electrophoresis analysis of isolates suggested that the strains colonizing the rectoanal junction were the same as those from the feces. Mucosal swab sampling was more sensitive than fecal sampling for determining the prevalence of E. coli O157 in feedlot cattle.

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

Escherichia coli O104:H4, an hybrid pathotype of Shiga toxigenic and enteroaggregative E. coli, involved in a major foodborne outbreak in Germany in 2011, has not been detected in cattle feces. Serogroup O104 with H type other than H4 has been reported to cause human illnesses, but their prevalence and characteristics in cattle have not been reported. Our objectives were to determine the prevalence of E. coli O104 in feces of feedlot cattle, by culture and PCR detection methods, and characterize the isolated strains. Rectal fecal samples from a total of 757 cattle originating from 29 feedlots were collected at a Midwest commercial slaughter plant. Fecal samples, enriched in E. coli broth, were subjected to culture and PCR methods of detection. The culture method involved immunomagnetic separation with O104-specific beads and plating on a selective chromogenic medium, followed by serogroup confirmation of pooled colonies by PCR. If pooled colonies were positive for the wzxO104 gene, then colonies were tested individually to identify wzxO104-positive serogroup and associated genes of the hybrid strains. Extracted DNA from feces were also tested by a multiplex PCR to detect wzxO104-positive serogroup and associated major genes of the O104 hybrid pathotype. Because wzxO104 has been shown to be present in E. coli O8/O9/O9a, wzxO104-positive isolates and extracted DNA from fecal samples were also tested by a PCR targeting wbdDO8/O9/O9a, a gene specific for E. coli O8/O9/O9a serogroups. Model-adjusted prevalence estimates of E. coli O104 (positive for wzxO104 and negative for wbdDO8/O9/O9a) at the feedlot level were 5.7% and 21.2%, and at the sample level were 0.5% and 25.9% by culture and PCR, respectively. The McNemar's test indicated that there was a significant difference (P < 0.01) between the proportions of samples that tested positive for wzxO104 and samples that were positive for wzxO104, but negative for wbdDO8/O9/O9a by PCR and culture methods. A total of 143 isolates, positive for the wzxO104, were obtained in pure culture from 146 positive fecal samples. Ninety-two of the 143 isolates (64.3%) also tested positive for the wbdDO8/O9/O9a, indicating that only 51 (35.7%) isolates truly belonged to the O104 serogroup (positive for wzxO104 and negative for wbdDO8/O9/O9a). All 51 isolates tested negative for eae, and 16 tested positive for stx1 gene of the subtype 1c. Thirteen of the 16 stx1-positive O104 isolates were from one feedlot. The predominant serotype was O104:H7. Pulsed-field gel electrophoresis analysis indicated that stx1-positive O104:H7 isolates had 62.4% homology to the German outbreak strain and 67.9% to 77.5% homology to human diarrheagenic O104:H7 strains. The 13 isolates obtained from the same feedlot were of the same PFGE subtype with 100% Dice similarity. Although cattle do not harbor the O104:H4 pathotype, they do harbor and shed Shiga toxigenic O104 in the feces and the predominant serotype was O104:H7.

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.

Shiga toxin-producing Escherichia coli (STEC), particularly O157, are major food borne pathogens. Non-O157 STEC, particularly O26, O45, O103, O111, O121, and O145, have also been recognized as a major public health concern. Unlike O157, detection procedures for non-O157 have not been fully developed. Our objective was to develop a multiplex PCR to distinguish O157 and the 'top six' non-O157 serogroups (O26, O45, O103, O111, O121, and O145) and evaluate the applicability of the multiplex PCR to detect the seven serogroups of E. coli in cattle feces. Published sequences of O-specific antigen coding genes, rfbE (O157) and wzx and wbqE-F (non-O157), were analyzed to design serogroup-specific primers. The specificity of amplifications was confirmed with 138 known STEC strains and the reaction yielded the expected amplicons for each serogroup. In feces spiked with pooled 7 STEC strains, the sensitivity of the detection was 4.1 × 10(5)CFU/g before enrichment and 2.3 × 10(2) after 6h enrichment in E. coli broth. Additionally, 216 fecal samples from cattle were collected and tested by multiplex PCR and cultural methods. The multiplex PCR revealed a high prevalence of all seven serogroups (178 [O26], 108 [O45], 149 [O103], 30 [O111], 103 [O121], 5 [O145], and 160 [O157]) of 216 samples in fecal samples. Cultural procedures identified 33.1% (53/160) and 35.5% (11/31) of PCR-positive samples for E. coli O157 and non-O157 serogroups, respectively. Samples that were culture-positive were all positive by the multiplex PCR. The multiplex PCR can be used to identify serogroups of putative STEC isolates.

To quantify associations at slaughter between Escherichia coli O157 carcass contamination, fecal-positive animals, and high-shedding animals within truckloads of finished cattle, we sampled up to 32 cattle from each of 50 truckloads arriving at a commercial abattoir in the Midwest United States during a 5-week summer period. Carcass swab samples collected pre-evisceration and fecal samples collected postevisceration were matched within animals and analyzed for the presence of E. coli O157, using enrichment, immunomagnetic separation, and plating on selective media (IMS). In addition, a direct plating procedure was performed on feces to identify high-shedding animals. E. coli O157 was isolated from 39 (2.6%) of 1,503 carcass samples in 15 (30%) truckloads, and 127 (8.5%) of 1,495 fecal samples in 37 (74%) truckloads. Fifty-five (3.7%) high-shedding animals were detected from 26 (52%) truckloads. Truckload high-shedder (Spearman rank-order correlation coefficient [r(s)] = 0.68), IMS-positive (r(s) = 0.48), and combined fecal (r(s) = 0.61) prevalence were significantly correlated with carcass prevalence. The probability of isolating E. coli O157 from a carcass was not significantly associated with the high-shedder or fecal IMS status of the animal from which the carcass was derived. However, the probability of carcass contamination was significantly associated with all truckload-level measures of fecal E. coli O157, particularly whether or not a high shedder was present within the truckload (odds ratio = 16.2; 95% confidence interval, 6.3-43.6). Our results suggest that high shedders within a truckload at slaughter could be a target for mitigation strategies to reduce the probability of preevisceration carcass contamination.

The rectoanal junction mucosal region is the site of colonization of Escherichia coli O157 in cattle. Our objective was to determine the genetic relatedness of E. coli O157 in the mucosa of the rectoanal junction to isolates from colon contents and feces. Colon contents and rectums were collected from cattle at harvest. Rectums were opened and feces were sampled with a cotton swab. The mucosa of the rectum was cleansed free of visible feces with water and saline. The region, 2 to 5 cm proximal to the rectoanal junction, was swabbed with a foam-tipped applicator and then incisions were made in this region and the submucosa was swabbed with an applicator. Isolation and identification of E. coli O157 was performed in accordance with well-documented methods. Prevalence of E. coli O157 in the colon contents, feces, rectal mucosa, and rectal submucosa was 21%, 29%, 54%, and 34%, respectively. Pulsed-field gel electrophoresis was used to compare clonal similarity among isolates from different sampling regions. Sixty-seven cattle had E. coli O157 isolated from the rectal mucosa swab and feces of which 82% were clonally similar (dice similarity95%) within animal. Escherichia coli O157 isolates from feces and colon contents were similar in 76% of cattle, but E. coli O157 isolates from the rectoanal mucosal swab and colon contents were only similar in 61.4% of cattle. Our results suggest that E. coli O157 in the feces may be from two sources, colonized in the rectoanal mucosa or transient in the gastrointestinal tract.

Culture-based methods to detect the six major non-O157 (O26, O45, O103, O111, O121 and O145) Shiga toxin-producing E. coli (STEC) are not well established. Our objectives of this study were to develop a culture-based method to detect the six non-O157 serogroups in cattle feces and compare the detection with a PCR method. Fecal samples (n = 576) were collected in a feedlot from 24 pens during a 12-week period and enriched in E. coli broth at 40° C for 6 h. Enriched samples were subjected to immunomagnetic separation, spread-plated onto a selective chromogenic medium, and initially pooled colonies, and subsequently, single colonies were tested by a multiplex PCR targeting six serogroups and four virulence genes, stx1, stx2, eae, and ehxA (culture method). Fecal suspensions, before and after enrichment, were also tested by a multiplex PCR targeting six serogroups and four virulence genes (PCR method). There was no difference in the proportions of fecal samples that tested positive (74.3 vs. 77.4%) for one or more of the six serogroups by either culture or the PCR method. However, each method detected one or more of the six serogroups in samples that were negative by the other method. Both culture method and PCR indicated that O26, O45, and O103 were the dominant serogroups. Higher proportions (P < 0.05) of fecal samples were positive for O26 (44.4 vs. 22.7%) and O121 (22.9 vs. 2.3%) serogroups by PCR than by the culture method. None of the fecal samples contained more than four serogroups. Only a small proportion of the six serogroups (23/640; 3.6%) isolated carried Shiga toxin genes. The culture method and the PCR method detected all six serogroups in samples negative by the other method, highlighting the importance of subjecting fecal samples to both methods for accurate detection of the six non-O157 STEC in cattle feces.