David G. Renter

Diversity, Frequency, and Persistence of Escherichia coli O157 Strains from Range Cattle Environments

ABSTRACT Genetic diversity, isolation frequency, and persistence were determined for Escherichia coli O157 strains from range cattle production environments. Over the 11-month study, analysis of 9,122 cattle fecal samples, 4,083 water source samples, and 521 wildlife fecal samples resulted in 263 isolates from 107 samples presumptively considered E. coli O157 as determined by culture and latex agglutination. Most isolates (90.1%) were confirmed to be E. coli O157 by PCR detection of intimin and Shiga toxin genes. Pulsed-field gel electrophoresis (PFGE) of XbaI-digested preparations revealed 79 unique patterns (XbaI-PFGE subtypes) from 235 typeable isolates confirmed to be E. coli O157. By analyzing up to three isolates per positive sample, we detected an average of 1.80 XbaI-PFGE subtypes per sample. Most XbaI-PFGE subtypes (54 subtypes) were identified only once, yet the seven most frequently isolated subtypes represented over one-half of the E. coli O157 isolates (124 of 235 isolates). Recurring XbaI-PFGE subtypes were recovered from samples on up to 10 sampling occasions and up to 10 months apart. Seven XbaI-PFGE subtypes were isolated from both cattle feces and water sources, and one of these also was isolated from the feces of a wild opossum (Didelphis sp.). The number of XbaI-PFGE subtypes, the variable frequency and persistence of subtypes, and the presence of identical subtypes in cattle feces, free-flowing water sources, and wildlife feces indicate that the complex molecular epidemiology of E. coli O157 previously described for confined cattle operations is also evident in extensively managed range cattle environments.

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in ...

ESCHERICHIA COLI O157:H7 IN FREE-RANGING DEER IN NEBRASKA

In order to determine the prevalence and distribution of the human pathogen, Escherichia coli O157:H7, in free-ranging deer, hunters were asked to collect and submit fecal samples from deer harvested during a regular firearm season (14–22 November 1998). Prior to the season, 47% of the hunters with permits in the southeastern Nebraska (USA) study area indicated a willingness to participate in the study. Approximately 25% of successful hunters in the area submitted deer fecal samples. Escherichia coli O157:H7 was cultured from four (0.25%) of 1,608 total samples submitted. All of the fecal samples that were properly identified (1,426) and all that were positive for E. coli O157:H7 were from white-tailed deer (Odocoileus virginianus). We were unable to detect a statistically significant geographic distribution pattern of E. coli O157:H7. The presence of E. coli O157:H7 in the feces of free-ranging deer has implications not only for hunters, consumers of venison, and others in contact with deer or deer feces, but also for the development of strategies aimed at reducing and/or controlling this pathogen in water sources and domestic livestock.

Effects of feeding wet corn distillers grains with solubles with or without monensin and tylosin on the prevalence and antimicrobial susceptibilities of fecal foodborne pathogenic and commensal bacteria in feedlot cattle.

Distillers grains, a coproduct of ethanol production from cereal grains, are composed principally of the bran, protein, and germ fractions and are commonly supplemented in ruminant diets. The objective of this study was to assess the effect of feeding wet distillers grains with solubles (WDGS) and monensin and tylosin on the prevalence and antimicrobial susceptibilities of fecal foodborne and commensal bacteria in feedlot cattle. Cattle were fed 0 or 25% WDGS in steam-flaked corn-based diets with the addition of no antimicrobials, monensin, or monensin and tylosin. Fecal samples were collected from each animal (n = 370) on d 122 and 136 of the 150-d finishing period and cultured for Escherichia coli O157. Fecal samples were also pooled by pen (n = 54) and cultured for E. coli O157, Salmonella, commensal E. coli, and Enterococcus species. Antimicrobial resistance was assessed by determining antimicrobial susceptibilities of pen bacterial isolates and quantifying antimicrobial resistance genes in fecal samples by real-time PCR. Individual animal prevalence of E. coli O157 in feces collected from cattle fed WDGS was greater (P < 0.001) compared with cattle not fed WDGS on d 122 but not on d 136. There were no treatment effects on the prevalence of E. coli O157 or Salmonella spp. in pooled fecal samples. Antimicrobial susceptibility results showed Enterococcus isolates from cattle fed monensin or monensin and tylosin had greater levels of resistance toward macrolides (P = 0.01). There was no effect of diet or antimicrobials on concentrations of 2 antimicrobial resistance genes, ermB or tetM, in fecal samples. Results from this study indicate that WDGS may have an effect on the prevalence of E. coli O157 and the concentration of selected antimicrobial resistance genes, but does not appear to affect antimicrobial susceptibility patterns in Enterococcus and generic E. coli isolates.

Enterohemorrhagic Escherichia coli O157: epidemiology and ecology in bovine production environments

Abstract Enterohemorrhagic Escherichia coli, particularly the O157(:H7) serogroup, has become a worldwide public health concern. Since cattle feces are often implicated as the source of E. coli O157 in human infections, considerable resources have been devoted to defining the epidemiology and ecology of E. coli O157 in cattle environments so that control might begin at the farm level. Diagnostic limitations and the complexity of often interrelated microbial, animal, herd, environmental and production factors have hindered the determination of the epidemiology, ecology and subsequent farm-level control of E. coli O157. The widespread distribution of E. coli O157, the transitory nature of fecal shedding, multiple potential environmental sources, lack of species specificity, and age-, feed- and time-related differences in cattle prevalence are documented. However, the significance and/or role of these factors in the epidemiology and ecology of E. coli O157 is still unclear. Cattle are a major source of E. coli O157, but it may be simplistic to believe that most herds are relatively closed systems with small percentages of cattle serving as true reservoirs. Practical on-farm control may require explicit definitions of the seemingly complex system(s) and the microbial, animal, herd, environmental and production factors involved in the multiplication, maintenance and transmission of E. coli O157.

Effects of Dried Distillers' Grain on Fecal Prevalence and Growth of Escherichia coli O157 in Batch Culture Fermentations from Cattle†

ABSTRACT Distillers’ grains (DG), a by-product of ethanol production, are fed to cattle. Associations between Escherichia coli O157 prevalence and feeding of DG were investigated in feedlot cattle (n = 379) given one of three diets: steam-flaked corn (SFC) and 15% corn silage with 0 or 25% dried distillers’ grains (DDG) or SFC with 5% corn silage and 25% DDG. Ten fecal samples were collected from each pen weekly for 12 weeks to isolate E. coli O157. Cattle fed 25% DDG with 5 or 15% silage had a higher (P = 0.01) prevalence of E. coli O157 than cattle fed a diet without DDG. Batch culture ruminal or fecal microbial fermentations were conducted to evaluate the effect of DDG on E. coli O157 growth. The first study utilized microbial inocula from steers fed SFC or dry-rolled corn with 0 or 25% DDG and included their diet as the substrate. Ruminal microbial fermentations from steers fed DDG had higher E. coli O157 contents than ruminal microbial fermentations from steers fed no DDG (P < 0.05) when no substrate was included. Fecal fermentations showed no DDG effect on E. coli O157 growth. In the second study with DDG as a substrate, ruminal fermentations with 0.5 g DDG had higher (P < 0.01) E. coli O157 concentrations at 24 h than ruminal fermentations with 0, 1, or 2 g DDG. In fecal fermentations, 2 g DDG resulted in a higher concentration (P < 0.05) at 24 h than 0, 0.5, or 1 g DDG. The results indicate that there is a positive association between DDG and E. coli O157 in cattle, and the findings should have important ramifications for food safety.

Serial evaluation of physiologic, pathological, and behavioral changes related to disease progression of experimentally induced Mannheimia haemolytica pneumonia in postweaned calves

OBJECTIVE To determine the usefulness of physiologic, behavioral, and pathological changes as objective indicators of early respiratory disease in calves with Mannheimia haemolytica pneumonia. ANIMALS 14 crossbred beef steers. PROCEDURES Disease was experimentally induced in healthy calves through endoscopic pulmonary inoculation of M haemolytica. Calves were necropsied on days 1, 2, 3, 5, 7, and 9 after inoculation. Physical examination variables (rectal temperature, heart rate, and respiration characteristics), clinical illness score, and degree of activity were assessed 3 times daily beginning 4 days prior to inoculation and continuing throughout the study. Twice before inoculation and on days 1, 2, 3, 5, 7, and 9, arterial blood gas measurements, serum biochemical analyses, and CBCs were performed. Pedometers and accelerometers were used to monitor cattle behavior and activity throughout the trial. RESULTS All calves became clinically ill after inoculation and had gross and histopathologic signs of bronchopneumonia. No variable was a reliable indicator of disease progression as judged by percentage of pulmonary involvement. However, activity as measured by total steps taken in a 24-hour period was lower after versus before disease induction. CONCLUSIONS AND CLINICAL RELEVANCE This single-pathogen challenge model successfully yielded clinical signs and pathological effects consistent with naturally acquired respiratory disease. Routine laboratory variables and subjective measures were not reliable indicators of lung involvement or the progression of pneumonia. However, activity, objectively measured with pedometers and accelerometers, appeared to be a promising indicator for early recognition of bovine respiratory disease.

Efficacy of a vaccine and a direct-fed microbial against fecal shedding of Escherichia coli O157:H7 in a randomized pen-level field trial of commercial feedlot cattle

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% distillers 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% distillers grains.

Prevalence and Serovars of Salmonella in the Feces of Free-Ranging White-Tailed Deer (Odocoileus virginianus) in Nebraska

To determine the prevalence and serovars of Salmonella in free-ranging deer, we cultured feces from white-tailed deer (Odocoileus virginianus) harvested by hunters during a regular firearm season in southeastern Nebraska (USA). We recovered Salmonella from 5 (1%; 95% confidence interval: 0.37–2.20%) of 500 samples and identified four different Salmonella enterica serovars [Litch-field (1), Dessau (1), Infantis (2), and Enteritidis (1)]. Although the prevalence of Salmonella in free-ranging deer appears to be low, the serovars recovered are known to be pathogenic to humans and animals.

Associations between the Presence and Magnitude of Escherichia coli O157 in Feces at Harvest and Contamination of Preintervention Beef Carcasses

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.