Effect of waste milk pasteurization on fecal shedding of Salmonella in preweaned calves.

The objective of the current research was to determine if pasteurization of nonsaleable waste milk influences fecal Salmonella concentrations and prevalence, or antimicrobial susceptibility and serotype of the cultured isolates. Holstein dairy calves (n = 211) were housed on a single commercial dairy in the southwestern United States and randomly allotted to be fed either pasteurized (PWM; n = 128 calves) or nonpasteurized waste milk (NPWM; n = 83 calves). Fecal samples were collected via rectal palpation or from freshly voided, undisturbed fecal pats, weekly during the first 4 wk of the animal's life and then again at weaning. Eight total collections were made and 1,117 fecal samples cultured for Salmonella. One isolate from each culture-positive fecal sample was preserved for antimicrobial susceptibility screening and serotyping. Sixty-nine percent of the fecal samples were culture positive for Salmonella with no difference due to treatment (67.7 and 69% Salmonella positive for PWM and NPWM treatments, respectively). Few fecal samples (178/1,117; 15.9%) contained Salmonella concentrations above the limit of detection (∼1 cfu/g of feces) with concentrations ranging from 1.0 to 6.46 cfu (log10)/g of feces. Concentration was not affected by treatment. Seventeen different serotypes were identified, the majority of which were Montevideo and Anatum. A greater percentage of Typhimurium (87 vs. 13%), Muenchen (88 vs. 12%), and Derby (91 vs. 9%) were recovered from calves fed PWM compared with NPWM-fed calves. Conversely, Newport (12.5 vs. 86%), Bredeney (22.2 vs. 77.8%), and Muenster (12.5 vs. 87.5%) were lower in PWM compared with NPWM treatments. The majority (66.7%) of isolates were susceptible to all of the antibiotics examined. Results from this one commercial dairy suggest that milkborne Salmonella is not an important vector of transmission in dairy neonates, nor does pasteurization of waste milk influence fecal shedding of this pathogen. Caution should be used, however, when extrapolating results to other farms as Salmonella contamination of milk on farm is well documented. The potential benefits of pasteurization in disease prevention outweigh the potential risks of feeding a nonpasteurized product and warrants incorporation into any calf-rearing program using nonsaleable waste milk for feeding young dairy neonates.

Evidence supporting vertical transmission of Salmonella in dairy cattle.

We set out to investigate whether Salmonella enterica could be recovered from various tissues of viable neonatal calves immediately following parturition. Eleven samples were aseptically collected from each of 20 calves and consisted of both left and right subiliac and prescapular lymph nodes (LN), mesenteric LN, spleen and liver, as well as intestinal tissue (including luminal contents) from the small intestine, caecum, spiral colon and rectum. In addition, a faecal sample was collected from 19 of the dams. Salmonella was recovered from at least one sample from 10 of the 20 neonates. Across all calves, Salmonella was recovered from 12·7% of all samples and from LN in particular, Salmonella was recovered from 10·0%, 5·0%, and 5·0% of subiliac, prescapular, and mesenteric LN, respectively. Within calves, Salmonella was recovered from 0% to 73% of samples and across tissues, estimates of Salmonella prevalence were greatest in the caecum (30%) but was never recovered from the right pre-scapular LN. These data provide evidence of vertical transmission from a dam to her fetus such that viable calves are born already infected and thereby not requiring faecal-oral exposure for transmission. This new knowledge ought to challenge - or at least add to - existing paradigms of Salmonella transmission dynamics within cattle herds.

Competitive effect of commensal faecal bacteria from growing swine fed chlortetracycline-supplemented feed on ß-haemolytic Escherichia coli strains with multiple antimicrobial resistance plasmids.

AIMS: To determine the differences in competitive fitness among Escherichia coli strains with different plasmid profiles when grown in suspension with commensal faecal bacteria from growing swine fed chlortetracycline-supplemented or unsupplemented diets. METHODS AND RESULTS: Five multiple drug-resistant (MDR) E. coli strains that possessed 0, 2, 6 or 8 plasmids were inoculated into anoxic faecal cultures from swine fed an unsupplemented (control) or chlortetracycline (50 g ton(-1))-supplemented (experimental) diet. On days 21 of chlortetracycline supplementation, faecal growth competition studies were performed. MDR E. coli were enumerated at 0, 6 and 24 h. The plasmid-free strain was below culturable limits in both the control and experimental cultures by 24 h. For each plasmid-bearing strain, there was no statistically significant difference in population CFU ml(-1) (P < 0.05) between the control and experimental cultures. CONCLUSIONS: There was no significant effect on the faecal microflora, owing to the inclusion of chlortetracycline, in the swine diets, that affected the growth of E. coli in the competition studies employed. Furthermore, these results suggest that the cost of maintaining plasmids in these E. coli strains had little influence on survivability. SIGNIFICANCE AND IMPACT OF STUDY: Mutations that led to antimicrobial resistance may have a greater impact on survivability than multiple plasmid carriage.

Prevalence and Antimicrobial Resistance of Nontyphoidal Salmonella enterica from Head Meat and Trim for Ground Product at Pork Processing Facilities.

ABSTRACT: Pork head meat may harbor Salmonella and contaminate other carcass by-products during harvest and fabrication. A large pork processing plant in the United States was sampled bimonthly for 11 months to determine the concentration, prevalence, seasonality, serotype diversity, and antimicrobial susceptibility of Salmonella enterica isolated from cheek meat and head trim of swine carcasses. Each collection consisted of 25 samples on two consecutive days in the morning and afternoon shifts, for a total of 100 cheek meat and 100 head trim samples each month. Tissues were cultured for Salmonella by using restrictive media and enrichment techniques, and a subset of isolates was serotyped, analyzed for antimicrobial susceptibility, and genome sequenced. Salmonella postenrichment prevalence did not differ (P = 0.20) between cheek meat (63%) and head trim (66%). Postenrichment prevalence differed (P < 0.05) by month (January, 94%; March, 80%; May, 54%; July, 59%; September, 47%; and November, 55%) and by processing shift (morning, 68%; afternoon, 62%). The subset (n = 618) of isolates selected for serotyping yielded 21 distinct serotypes: Typhimurium (49%), Infantis (10%), Heidelberg (8%), I 4,[5],12:i:- (8%), and 17 other types (≤5%). In total, 407 multidrug-resistant (MDR; resistance to three or more antibiotic classes) isolates were identified. There were 120 isolates that exhibited the penta-resistant ACSSuT phenotype. In addition, 113 isolates exhibited decreased susceptibility to ciprofloxacin (MIC ≥ 0.12 µg/mL). Resistance genes blaCARB, blaSHV, blaTEM, aac(6')-Ib-cr,qnrB, sul2, and dfrA were expressed in numerous MDR Salmonella isolates. The data herein suggest that pork products from the head, compared with data reported for carcasses, may have a relatively high prevalence of Salmonella with diverse serotypes and MDR.

Reduction of multidrug-resistant and drug-susceptible Salmonella in ground beef and freshly harvested beef briskets after exposure to commonly used industry antimicrobial interventions.

Two separate studies were conducted to examine the differences in survivability of multidrug-resistant (MDR) and drug-susceptible Salmonella in fresh meats in a simulated industry environment. Beef trim from a commercial facility was inoculated with either MDR (AmpC phenotype) or drug-susceptible Salmonella (SUS) cocktails (10(6) CFU/ml). Antimicrobial interventions included 3% lactic acid (LA), 1,000 ppm of acidified sodium chloride (ASC), ambient water (AW), and an inoculated control with no intervention (CTRL). Each aliquot was ground and formed into patties and packaged using high-O(2) modified atmosphere packaging. Samples for microbiological evaluation were collected on days 0, 7, 10, and 14. In the second study, beef briskets were collected immediately after harvest. Inoculation and antimicrobial application were the same, except treatments were heated and there was an additional hot water treatment. All beef briskets were refrigerated, and samples were collected at 0, 6, and 24 h. For the first study, the overall effectiveness of the treatments (from most effective to least effective) was LA, ASC, CTRL, and AW. Significant differences were observed only between MDR and SUS Salmonella when AW was applied (P = 0.02), and bacterial loads with AW were significantly greater (P < 0.01) for MDR Salmonella. In the second study, the intervention effectiveness ranked LA, ASC, hot water, AW, and CTRL. Significant differences between MDR and SUS Salmonella levels were not detected for any intervention or sampling time point. These data indicate that MDR and SUS variants of Salmonella behave similarly in response to the antagonistic action of antimicrobials commonly used in beef facilities.

Diversity and susceptibility of Enterococcus isolated from cattle before and after harvest.

To investigate evidence of cross-contamination and to determine patterns of antimicrobial drug susceptibility of Enterococcus isolates in a commercial cattle processing system, samples were collected from 60 cattle shipped to a commercial abattoir. Enterococcus isolates were recovered from fecal and hide samples collected immediately before shipment from a feedlot to the abattoir, from postexsanguination hide samples at the abattoir, and from carcass samples collected after hide removal (preevisceration) and in the cooler. Of the fecal samples, 53.9% were culture positive for Enterococcus. Of hide samples collected at the feedlot, 77.8% were positive for Enterococcus, significantly lower (P < 0.01) than the proportion of hides that were culture positive at the abattoir (96.1%). For preevisceration carcass samples, Enterococcus was recovered from 58.3% of carcasses. Only 8.3% of the carcasses sampled in the cooler yielded Enterococcus. Resistance among Enterococcus isolates was common regardless of the type or location of sample from which the isolate was recovered. All 279 Enterococcus isolates were resistant to at least one antimicrobial drug, and 179 (64.2%) of these isolates were resistant to at least six drugs. The most common resistance was to chloramphenicol (100% of isolates) followed by flavomycin (90.3%), lincomycin (87.8%), tylosin (78.5%), erythromycin (76.3%), tetracycline (58.9%), quinupristin-dalfopristin (47.7%), bacitracin (17.9), streptomycin (9.0%), ciprofloxacin (1.4%), linezolid (0.7%), and salinomycin (0.4%). Enterococcus isolates also were characterized using pulsed-field gel electrophoresis to evaluate molecular similarities. Similar or indistinguishable electrophoresis patterns were found among isolates recovered at the feedlot and in the plant, providing evidence that feedlot-origin bacterial isolates are being transferred from cattle to carcasses within the processing environment through cross-contamination.

Effects of a siderophore receptor and porin proteins-based vaccination on fecal shedding of Escherichia coli O157:H7 in experimentally inoculated cattle.

The efficacy of a vaccine containing outer membrane siderophore receptor and porin (SRP) proteins for reducing fecal prevalence and shedding of Escherichia coli O157:H7 was evaluated in cattle inoculated with E. coli O157:H7. Thirty calves were randomly assigned to one of two groups, and on days 1 and 21 these calves were given subcutaneous injections of either a placebo (control) or the vaccine. Blood was collected weekly to monitor the serum anti-SRP antibody titers. Two weeks after the second vaccination, calves were orally inoculated with a mixture of five strains of nalidixic acid-resistant (NalR) E. coli O157:H7. Fecal samples and rectoanal mucosal swabs were collected daily for the first 5 days and then three times each week for the following 4 weeks to determine the presence and enumerate the fecal concentration of NalR E. coli O157:H7. At necropsy on day 35, gut contents and tissue swabs were collected to determine the presence and concentration of NalR E. coli O157:H7. Vaccinated cattle had significantly higher anti-SRP antibody titers than did control cattle, with a significant treatment x week interaction (P < 0.01). Vaccination of cattle with the SRP protein tended to decrease fecal concentration (1.9 versus 1.6 log CFU/g) of NalR E. coli O157:H7 (P = 0.10). The number of calves that were fecal culture positive for E. coli O157:H7 was lower (P = 0.05) in the vaccinated group than in the control group. The E. coli O157:H7 SRP vaccine tended to reduce fecal prevalence and concentration of E. coli O157:H7 in cattle orally inoculated with NalR E. coli 0157:H7 and may be a useful prehavest intervention strategy. Future research must be conducted on natural prevalence in feedlot operations to further evaluate the efficacy of this novel vaccine.

Quantification of the bla(CMY-2) in feces from beef feedlot cattle administered three different doses of ceftiofur in a longitudinal controlled field trial.

The objective of this longitudinal controlled trial was to quantitatively compare carriage of a gene encoding for ceftiofur-resistance (bla(CMY-2)), standardized to a reference gene (16SrRNA), among total community DNA extracted from fecal samples collected from cattle treated with three different dose regimens of ceftiofur crystalline-free acid (CCFA) versus those untreated (controls). Sixty-one steers were assigned to three treatment regimens and housed in six pens. In each pen, five steers were treated and five were controls (one of the pens had six controls). CCFA administration was as follows: two-thirds dose treatment (4.4 mg/kg, on day 0), single-dose treatment (6.6 mg/kg, on day 0), and three-dose treatment (6.6 mg/kg, on days 0, 6, and 13). Fecal samples were collected on days 0, 3, 7, 10, 14, 18, 21, and 28. The gene copy numbers/gram of feces for bla(CMY-2) and 16SrRNA were determined in total community DNA samples using quantitative real-time PCR. The relationships between the quantities of standardized bla(CMY-2), nonstandardized bla(CMY-2), and nonstandardized 16SrRNA, and the explanatory variables (treatment, time, and treatment x time) were assessed using repeated measures mixed models. There were significant differences in each of the three models with respect to each explanatory variable. Overall, while steers administered three doses and two-thirds dose of CCFA had significantly higher quantities of nonstandardized bla(CMY-2) than controls, the standardized values were lower. The administration of CCFA in feedlot cattle may provide selection pressure favoring higher levels of bla(CMY-2) carriage, but this may also lead to concurrent reductions in the total bacterial population (as reflected by lowered 16SrRNA) during the treatment period.

An exploratory observational study to develop an improved method for quantifying beef carcass salable meat yield.

Eighty-seven grain-finished steers were harvested, evaluated, and fabricated into wholesale cuts to determine what measured composition indicators most accurately describe the percentage of closely trimmed salable meat yield. Indicators of lean and fat composition present at the cross-section between the 12th and 13th ribs were objectively evaluated using Assess image analysis software. Salable meat yield ranged from 50.18% to 72.92%, trimmable fat yield ranged from 12.87% to 36.69%, and bone yield ranged from 10.07% to 19.21%. Regression models were developed to estimate percentage of total salable meat yield. Composition indicators chosen to predict salable meat yield included hot carcass weight (HCW), perinephric fat weight, longissimus muscle area (LMA), subcutaneous fat thickness (SFT), ratio of LMA to subcutaneous fat area, and ratio of subcutaneous fat depth to HCW. These results indicate that prediction of beef carcass salable meat yield can be improved via modification to current measures used in the USDA yield grade equation and addition of new measures.

Environmental dust exposure as a factor contributing to an increase in Escherichia coli O157 and Salmonella populations on cattle hides in feedyards.

A study was conducted to determine the impact of exposure to dust in the cattle load-out area in feedyards on pathogen contamination of cattle hides. A total of 250 cattle hides were sampled during summer and fall months, which are associated with elevated prevalence of Escherichia coli O157 in West Texas. Animals were removed from their home pens and restrained in a chute and sampled prior to exposure to dust generated as a result of a simulated loading exercise. The cattle hides were sampled again after exposure to the loading dust to determine total numbers of pathogens on cattle hides on leaving their home pen (before loading) and on cattle hides after exposure to the dust in the loading area. Air and dirt samples from the home pens and the cattle load-out area were also collected. The presence of E. coli O157 and Salmonella was determined in all the samples, and when a positive sample was identified, the total numbers of these bacteria present were enumerated. The total numbers of pathogens increased after dust exposure; Salmonella counts increased from 1.09 log most probable number (MPN)/cm2 to 1.74 log MPN/cm2 after exposure, and E. coli O157 counts increased from 0.80 to 2.35 log MPN/cm2 after sampling. E. coli O157 and Salmonella were recovered from the air samples during dust generation at 6.66 and 11.1%, respectively. Salmonella and E. coli O157 prevalence was not changed and was not associated with the exposure to the dust. Results indicate airborne dust generated as a result of cattle movement and loading could be an important determining factor in total numbers of pathogens recovered on cattle hides.

Fecal shedding of foodborne pathogens by Florida-born heifers and steers in U.S. beef production segments.

The objective in this study was to assess breed effects in fecal prevalence of Escherichia coli O157:H7 in heifers on a development program in Florida and in their steer half siblings in stocker and feedlot phases in Oklahoma. A secondary objective was to characterize fecal shedding of Campylobacter and Salmonella in subsets of the same samples. After weaning, heifers (n = 501; purebreds and F1 crosses of Angus, Brahman, and Romosinuano) were preconditioned and placed in a local development program. Steers (n = 481) were transported to Oklahoma, where they grazed wheat for 6 months and then were placed in feedlot pens. Fecal samples were obtained at least every 28 days for 12 months on most animals. None of the 10,982 samples tested positive for E. coli O157:H7. Overall fecal prevalences of Campylobacter and Salmonella in heifers were 1.7 and 0.04%, respectively. Corresponding overall prevalences in steer samples were 27.2 and 0.6%. Campylobacter isolates were mostly C. jejuni and were tetracycline resistant. Eight Salmonella isolates were Salmonella Typhimurium that were either quad or penta resistant, most often to ampicillin, chloramphenicol, sulfamexathole, and tetracycline. Feedlot steers had greater odds of positive detection of Campylobacter (odds ratio, 8.5; confidence interval, 3.7, 19.5) than when grazing winter wheat. No breed effect was detected for fecal prevalence of these pathogens.

Molecular detection and quantification of viable probiotic strains in animal feedstuffs using the commercial direct fed microbial Lactobacillus animalis NP51 as a model.

Lactobacillus animalis NP51 is a direct-fed microbial strain (DFM) extensively used as a pre-harvest food safety mitigation in feedlot cattle due to its antagonistic effects against human foodborne pathogens such as Salmonella and Escherichia coli O157:H7. NP51 not only promotes overall gut health but interferes with the ability of these pathogens to colonize the gastrointestinal tract of cattle. As a result, NP51 reduces fecal shedding of Salmonella and E. coli O157:H7 in cattle presented for harvest and the load of these pathogens that enter the human food chain. Cattle are administered a high dose (1 × 109 CFU/head/day) of NP51 to reduce fecal shedding of foodborne pathogens. Ensiled animal feedstuffs naturally contain a high load of lactic acid bacteria (LAB) and it is not possible to detect and quantify the level of a specific LAB strain (e.g., NP51) in this matrix using traditional microbiological culture. The purpose of this study was to develop a molecular method to detect and quantify viable populations of a specific LAB strain (e.g., NP51) in cattle feedstuffs. The NP51 whole genome sequence was aligned with closely related LAB clustering within the same well-supported clade in a LAB phylogeny derived from 30 conserved amino acid encoding sequence to identify orthologs. A sequence encoding recombinational DNA repair protein RecT was found to be unique to NP51 and used to design primers and a probe for molecular detection and quantification of NP51. The primers and probe were confirmed to be specific to NP51 in vitro. Total RNA was extracted from silage samples, including samples naturally inoculated in the field and control samples that were artificially spiked with a range of NP51 concentrations in the laboratory. Reverse-transcriptase quantitative real-time (RT-qRTi) PCR was used to quantify cDNA copies in samples and cycle threshold (Ct) values were compared to a standard curve to estimate NP51 concentrations. Our results indicate this novel molecular method is suitable to confirm the presence and estimate the concentration of a specific LAB strain in animal feedstuffs containing high background levels of LAB.

Reduction of Escherichia coli O157 and Salmonella in feces and on hides of feedlot cattle using various doses of a direct-fed microbial.

In this study, the effectiveness of direct-fed microbials at reducing Escherichia coli O157 and Salmonella in beef cattle was evaluated. Steers (n=240) received one of the following four treatment concentrations: control = lactose carrier only; low = 1 X 10(7) CFU per steer daily Lactobacillus acidophilus NP51; medium = 5 x 10(8) CFU per steer daily L. acidophilus NP51; and high = 1 x 10(9) CFU per steer daily L. acidophilus NP51. Low, medium, and high diets also included 1 x 10(9) CFU per steer Propionibacterium freudenreichii NP24. Feces were collected from each animal at allocation of treatment and found to have no variation (P = 0.54) between cohorts concerning E. coli O157 recovery. Feces and hide swabs were collected at harvest and analyzed for the presence of E. coli O157 by immunomagnetic separation and Salmonella by PCR. No significant dosing effects were detected for E. coli O157 recovery from feces at the medium dose or from hides at the medium and high doses. E. coli O157 was 74% (P < 0.01) and 69% (P < 0.01) less likely to be recovered in feces from animals receiving the high and low diets, respectively, compared with controls. Compared with controls, E. coli O157 was 74% (P = 0.05) less likely to be isolated on hides of cattle receiving the low dose. No significant dosing effects were detected for Salmonella recovery from feces at the medium and low doses or from hides at any doses. Compared with controls, Salmonella was 48% (P = 0.09) less likely to be shed in feces of cattle receiving the high dose. No obvious dose-response of L. acidophilus NP51 on recovery of E. coli O157 or Salmonella was detected in our study.

Prevalence and enumeration of Escherichia coli O157 in steers receiving various strains of Lactobacillus-based direct-fed microbials.

The objective of this research was to evaluate the effect of daily dietary inclusion of specific strains of Lactobacillus acidophilus on prevalence and concentration of Escherichia coli O157 in harvest-ready feedlot cattle. Five hundred yearling steers were housed in pens of 10 animals each. At arrival, steers were randomly allocated to one of five cohorts. Four of the cohorts were fed various strains and dosages of Lactobacillus-based direct-fed microbials throughout the feeding period. Fecal samples were collected from the rectum of each animal immediately prior to shipment to the abattoir. E. coli O157 was detected using selective enrichment and immunomagnetic separation techniques. For positive samples, E. coli O157 concentration was estimated using a most-probable-number (MPN) technique that included immunomagnetic separation. Prevalence varied among the cohorts (P < 0.01). The prevalence in the controls (26.3%) was greater (P < 0.05) than that in cattle supplemented with L. acidophilus strains NP51, NP28, or NP51-NP35 (13.0, 11.0, and 11.0%, respectively). The greatest E. coli O157 concentration was also observed in the controls (3.2 log MPN/g of feces); this concentration was greater (P < 0.05) than that observed in positive animals receiving NP51, NP28, or NP51-NP35 (0.9, 1.1, 1.7 log MPN/g of feces, respectively). Specific strains of Lactobacillus-based direct-fed microbials effectively reduced the prevalence and concentration of E. coli O157 in harvest-ready cattle, whereas others did not. When using direct-fed microbials to reduce carriage of E. coli O157 in cattle, it is important to select appropriately validated products.

Comparison of rapid enzyme-linked immunosorbent assay and immunomagnetic separation methods for detection of Escherichia coli O157 in fecal, hide, carcass, and ground beef samples.

Rapid enzyme-linked immunosorbent assays (ELISAs) are approved for detection of Escherichia coil O157 in beef products. However, these kits have also been used in the industry to detect this pathogen on hides or in feces of cattle, although this use has not been validated. The objective of this study was to compare commercially available ELISAs (E. coli Now, Reveal, and VIP) with immunomagnetic separation along with selective media to detect E. coli O157 on hides, in feces, and in medium- and low-level-inoculated ground beef and carcasses (simulated by using briskets) samples. Naturally infected hide and fecal samples were subjected to both the immunomagnetic separation method and ELISAs for the detection of E. coli O157. Additionally, E. coli O157 inoculated and noninoculated ground beef and beef briskets were used to simulate meat and carcass samples. When comparing the detection results from the ELISAs (E. coli Now, Reveal, and VIP) to the immunomagnetic separation method, poor agreement was observed for fecal samples (kappa = 0.10, 0.02, and 0.03 for E. coli Now, Reveal, and VIP, respectively), and fair-to-moderate agreement was observed for hide samples (kappa = 0.30, 0.51, and 0.29 for E. coli Now, Reveal, and VIP, respectively). However, there was near-perfect agreement between the immunomagnetic separation method and ELISAs for ground beef (kappa = 1, 1, and 0.80 for E. coli Now, Reveal, and VIP, respectively) and brisket (kappa = 1, 1, and 1 for E. coli Now, Reveal, and VIP, respectively) samples. Assuming immunomagnetic separation is the best available method, these data suggest that the ELISAs are not useful in detecting E. coli O157 from hide or fecal samples. However, when ELISAs are used on ground beef and beef brisket samples they can be used with a high degree of confidence.

Development and validation of a most-probable-numberimmunomagnetic separation methodology of enumerating Escherichia coli O157 in cattle feces.

A method to validate enumeration of Escherichia coli O157 in fecal samples from feedlot cattle was developed in these studies. Due to background flora, bovine fecal sample enumeration cannot be performed by simple direct plating techniques. Known quantities of E. coli O157:H7 were inoculated into feces, and populations were determined by direct plating of the cocktail (studies 1, 2, and 3) and manure and cocktail (studies 4 and 5) mixtures and compared with a most-probable-number (MPN)-immunomagnetic separation (IMS) method. The three-tube MPN combined preenrichment in gram-negative broth with confirmation using IMS. Five separate enumeration studies (study 1, sterile feces inoculated with 10(2) E. coli O157:H7 per g; study 2, nonsterile feces inoculated with 10(3) E. coli O157:H7 per g; study 3, nonsterile feces inoculated with 10(1) E. coli O157:H7 per g; study 4, sterile feces inoculated with 10(4) streptomycin-resistant E. coli O157:H7 per g; and study 5, sterile feces inoculated with 10(2) streptomycin-resistant E. coli O157:H7 per g) were conducted. These studies were performed to determine the precision, accuracy, and specificity at low and high levels of pathogen contamination in feces, using direct plating compared with the MPN-IMS methodology tested. There was an overall difference (P < 0.01) between direct plating and MPN-IMS methodologies, but this difference was biologically negligible due to the difference in least-squares means (0.29 +/- 0.10) being so low. The direct plating and MPN-IMS methods were correlated (r = 0.93). These results suggest that using the MPN-IMS procedures is an effective method of estimating E. coli O157 populations in naturally infected bovine fecal samples.

Distribution of Escherichia coli 0157 and Salmonella on hide surfaces, the oral cavity, and in feces of feedlot cattle.

To determine the distribution of pathogens on cattle hides at the feedlot, samples were collected from six hide surface locations (back, flank, hock, neck, perineum, and ventrum), the oral cavity, the rectal-anal junction, and the feces of feedlot cattle and subjected to Escherichia coli 0157 detection via culture methods and to Salmonella detection via PCR. E. coli 0157 was isolated from one or more of the sampling locations from 31 (42.5%) of the 73 animals sampled. Location-specific prevalence of E. coli 0157 was 5% for back samples, 5% for flank samples, 12% for hock samples, 7% for neck samples, 12% for perineum samples, 8% for ventrum samples, 1% for oral cavity samples, 4% for rectal-anal junction swabs, and 23% for fecal grab samples. Salmonella was isolated from one or more of these sample locations from 100% (50 of 50 samples) of all animals sampled. Location-specific prevalence of Salmonella was 76% for back samples, 74% for flank samples, 94% for hock samples, 76% for neck samples, 88% for perineum samples, 86% for ventrum samples, 94% for oral cavity samples, 64% for rectal-anal junction swabs, and 50% for fecal grab samples. The sampling locations that maximized the likelihood of finding E. coli 0157 and Salmonella (84 and 96%, respectively) if the animal was positive at one sampling location or more were the hock, perineum, and fecal grab. These data suggest that the use of multiple sample locations is useful when isolating these pathogens from feedlot cattle. Focusing on one sampling location may underestimate the prevalence.

Molecular detection assay of five Salmonella serotypes of public interest: Typhimurium, Enteritidis, Newport, Heidelberg, and Hadar.

Foodborne illnesses due to Salmonella represent an important public-health concern worldwide. In the United States, a majority of Salmonella infections are associated with a small number of serotypes. Furthermore, some serotypes that are overrepresented among human disease are also associated with multi-drug resistance phenotypes. Rapid detection of serotypes of public-health concern might help reduce the burden of salmonellosis cases and limit exposure to multi-drug resistant Salmonella. We developed a two-step real-time PCR-based rapid method for the identification and detection of five Salmonella serotypes that are either overrepresented in human disease or frequently associated with multi-drug resistance, including serotypes Enteritidis, Typhimurium, Newport, Hadar, and Heidelberg. Two sets of four markers were developed to detect and differentiate the five serotypes. The first set of markers was developed as a screening step to detect the five serotypes; whereas, the second set was used to further distinguish serotypes Heidelberg, Newport and Hadar. The utilization of these markers on a two-step investigation strategy provides a diagnostic specificity of 97% for the detection of Typhimurium, Enteritidis, Heidelberg, Infantis, Newport and Hadar. The diagnostic sensitivity of the detection makers is >96%. The availability of this two-step rapid method will facilitate specific detection of Salmonella serotypes that contribute to a significant proportion of human disease and carry antimicrobial resistance.

Twenty-four hour Holter monitoring in finishing cattle housed outdoors.

INTRODUCTION: Atrial premature complexes have been reported to be the most common arrhythmia in cattle and is suspected to be secondary to systemic disease, especially gastrointestinal disease. In order to properly identify pathologic arrhythmia in cattle, the normal rhythm and arrhythmia prevalence should be defined. The objective of this study was to determine the normal heart rate, rhythm, number of ventricular premature complexes (VPCs), and atrial premature complexes (APCs) in unrestrained Angus steers. ANIMALS: Twenty-seven client owned steers with unremarkable physical examinations and serum biochemical analyses were used. MATERIALS AND METHODS: Twenty-four hour Holter monitors, attached by a custom-made harness, were retrospectively evaluated. Three lead electrocardiographic registrations of good quality and normal sinus rhythm were obtained from all steers in the study. RESULTS: The mean heart rate was 66.8 bpm ± 16.4 bpm. Ventricular premature complexes were rare (noted in 14.8% of steers), and APCs were common (noted in 85% of the steers). Simple second degree AV block was observed in 18.5% of the steers. CONCLUSION: In summary, healthy steers have rare single VPCs, although it is possible for an individual animal to have apparent more frequent VPCs. Mean heart rate varies with a diurnal pattern similar to other species. Atrial premature complexes are the most prevalent abnormality observed in feedlot steers.

Transferability of antimicrobial resistance from multidrug-resistant Escherichia coli isolated from cattle in the USA to E. coli and Salmonella Newport recipients.

OBJECTIVES: This study aimed to evaluate conjugative transfer of cephalosporin resistance among 100 strains of multidrug-resistant Escherichia coli (MDRE) to Salmonella enterica serotype Newport and E. coli DH5α recipients. METHODS: Phenotypic and genotypic profiles were determined for MDRE as well as for Salmonella Newport (trSN) and E. coli DH5α (trDH) transconjugants. RESULTS: Of 95 MDRE donor isolates, 26 (27%) and 27 (28%) transferred resistance to trSN and trDH recipients, respectively. A total of 27 MDRE (27%) were confirmed as extended-spectrum ß-lactamase (ESBL)-producers based on the double-disk synergy assay and whole-genome sequencing (WGS). WGS was performed on 25 of the ESBL-producing isolates, showing that 2 isolates carried blaCTX-M-6, 22 possessed blaCTX-M-32 and 1 was negative for blaCTX-M genes. Fourteen of the ESBLs sequenced were qnrB19. Differential transfer of IncA/C and IncN from MDRE32 was observed between trSN32 and trDH32. IncN-positive trDH32 displayed an ESBL phenotype, whereas IncA/C-positive trSN32 displayed an AmpC phenotype. The rate of ESBL transfer to trSN and trDH recipients was 11% and 96%, respectively. CONCLUSIONS: Twenty-seven MDRE were phenotypically identified as ESBL-producers. WGS of 25 MDRE revealed that 2 and 22 isolates carried blaCTX-M-6 and blaCTX-M-32, respectively. One multidrug-resistant isolate exhibited conversion from an AmpC phenotype to an ESBL phenotype with the transfer of only the IncN plasmid. The rate of resistance transfer to Salmonella or E. coli recipients was nearly identical. However, the ESBL phenotype was transferred with significantly greater prevalence to E. coli compared with Salmonella Newport (96% and 11%, respectively).