Guy H. Loneragan

Prevalence of Escherichia coli O157:H7 and performance by beef feedlot cattle given Lactobacillus direct-fed microbials.

Fecal shedding of Escherichia coli O157:H7, the prevalence of Escherichia coli O157:H7 in pens and on carcasses and hides, and cattle performance as a result of daily dietary supplementation with Lactobacillus-based direct-fed microbials (DFMs) were evaluated in a feeding trial involving 180 beef steers. Steers were evaluated for shedding of E. coli O157:H7 by an immunomagnetic separation technique on arrival at the feedlot, just before treatment with the DFMs, and every 14 days thereafter until slaughter. Composite pen fecal samples were collected every 14 days (alternating weeks with animal testing), and prevalence on hides and carcasses at slaughter was also evaluated. Feedlot performance (body weight gain and feed intake) was measured for the period during which the DFMs were fed. Gain efficiency was calculated as the ratio of weight gain to feed intake. Lactobacillus acidophilus NPC 747 decreased (P < 0.01) the shedding of E. coli O157:H7 in the feces of individual cattle during the feeding period. E. coli O157:H7 was approximately twice as likely to be detected in control animal samples as in samples from animals receiving L. acidophilus NPC 747. In addition, DFM supplementation decreased (P < 0.05) the number of E. coli O157:H7-positive hide samples at harvest and the number of pens testing positive for the pathogen. Body weight gains (on a live or carcass basis) and feed intakes during the DFM supplementation period did not differ among treatments. Gain efficiencies on a live-weight basis did not differ among treatments, but carcass-based gain/feed ratios tended (P < 0.06) to be better for animals receiving the two DFM treatments than for control animals. The results of this study suggest that the feeding of a Lactobacillus-based DFM to cattle will decrease, but not eliminate, fecal shedding of E. coli O157:H7, as well as contamination on hides, without detrimental effects on performance.

Reduction of Escherichia coli O157 in finishing beef cattle by various doses of Lactobacillus acidophilus in direct-fed microbials.

Our objective was to evaluate the effects of three doses of Lactobacillus acidophilus strain NP51 and a combination treatment of strains NP51 and NP45 on prevalence of Escherichia coli O157 in cattle. Three hundred steers were assigned randomly to 60 pens (five steers per pen) and received one of five treatments: (i) control, no added direct-fed microbial; (ii) HNP51, high dose of NP51 at 10(9) CFU per steer daily; (iii) MNP51, NP51 at 10(8) CFU per steer daily; (iv) LNP51, low dose of NP51 at 10(7) CFU per steer daily; and (v) NP51+45, NP51 at 10(9) CFU per steer daily and NP45 at 106 CFU per steer daily. All direct-fed microbial treatments included Propionibacterium freudenreichii at 10(9) CFU per steer. Individual rectal fecal samples were collected on arrival and every 28 days throughout the feeding period. Fecal and hide samples were collected on the day of harvest. Samples were analyzed for presence of E. coli O157 using immunomagnetic separation methods. Cattle receiving HNP51, MNP51, and LNP51 had a lower prevalence (P < 0.01) of E. coli O157 throughout the feeding period compared with the controls, and the dose response for NP51 was a linear decrease in prevalence with increasing dose (P < 0.01). No decrease in prevalence for cattle receiving the combination NP51+45 was detected compared with controls (P = 0.15). E. coli O157 prevalences averaged across collection times were 23.9, 10.5, 9.9, 6.8, and 17.3% for cattle in the control, LNP51, MNP51, HNP51, and NP51 +45 groups, respectively. Least squares mean estimates of fecal prevalence at harvest of E. coli O157 were 31.7, 12.5, 17.4, 8.2, and 41.6% among cattle in the control, LNP51, MNP51, HNP51, and NP51+45 groups, respectively. Least squares mean estimates of the percentage of positive hide samples at harvest were 8.7, 5.9, 4.8, 3.4, and 8.6% among cattle in the control, LNP51, MNP51, HNP51, and NP51+45 groups, respectively. The greatest decrease in E. coli O157 carriage was achieved using NP51 at 10(9) CFU per steer.

Dietary Supplementation with Lactobacillus- and Propionibacterium-Based Direct-Fed Microbials and Prevalence of Escherichia coli O157 in Beef Feedlot Cattle and on Hides at Harvest

The objective of this study was to describe the prevalence of Escherichia coli O157 in the feces and on the hides of finishing beef cattle fed a standard diet and those fed diets supplemented with direct-fed microbials. Two hundred forty steers received one of four treatments throughout the feeding period: (i) control: no added microbials; (ii) HNP51: high dose of Lactohacillius acidophilus strain NP 51 (10(9) CFU per steer daily) and Propionibacterium freudenreichii (10(9) CFU per steer daily); (iii) HNP51+45: high dose of NP 51 (10(9) CFU per steer daily), P. freudenreichii (10(9) CFU per steer daily), and L. acidophilus NP 45 (10(6) CFU per steer daily); or (iv) LNP51+45: low dose of NP 51 (10(6) CFU per steer daily), P. freudenreichii (10(9) CFU per steer daily), and NP 45 (10(6) CFU per steer daily). Samples were collected from each animal and analyzed for the presence of E. coli O157 using immunomagnetic separation methods on day 0 (feces), 7 days before harvest (feces), and at harvest (feces and hide). At the end of the feeding period, cattle receiving HNP51 were 57% less likely to shed detectable E. coli O157 in their feces than were the controls (P < 0.01). For animals receiving HNP51+45 and LNP51+45, fecal prevalence did not differ from that of the controls. The prevalence of positive hide samples was least among cattle receiving HNP51+45 (3.3%); these animals were 79% less likely (P < 0.06) to have a positive hide sample than were the controls (prevalence = 13.8%). There was poor agreement of the culture results between fecal and hide samples collected from the same animal (kappa = 0.08; confidence interval = -0.05 to 0.2). Cattle supplemented with a high dose of NP 51 had reduced E. coli O157 prevalence in both fecal and hide samples, indicating that this treatment may be an efficacious preharvest intervention strategy.

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.

Use of a Siderophore Receptor and Porin Proteins-Based Vaccine to Control the Burden of Escherichia coli O157:H7 in Feedlot Cattle

OBJECTIVE To quantify the efficacy of a siderophore receptor and porin (SRP) proteins-based vaccine on the burden of Escherichia coli O157:H7 in feedlot cattle. METHODS Two research trials were conducted in field conditions. In each trial, cattle (n = 1252 in trial #1; n = 1284 in trial #2) were systematically divided into 20 pens and were administered either the E. coli O157 SRP vaccine or a placebo (trial #1: days 0 and 21; trial #2: days 0, 21, and 42). Rectal fecal samples were collected on day 0, and pen floor samples were collected on days 21, 35, 70, and 85 for trial #1 and on days 42 and 98 for trial #2. On day 85 of trial #1, rectoanal mucosal swab samples and hide swab samples were collected. Cattle were weighed on days 0, 21, and 85 (trial #1) or 1 week before harvest (trial #2). RESULTS In trial #1, prevalence of E. coli in the feces was lower in vaccinates compared to control animals (p = 0.04). On day 85, the likelihood of an animal being positive at any site was less among vaccinates than controls (p = 0.02). In trail #2, vaccination with SRP was associated with reduced shedding by 85.2% on day 98 (p < 0.01) but not on days 0 or 42. Vaccination with SRP was associated with a 98.2% reduction in concentration of E. coli O157 in fecal samples (2.54 vs. 0.80 log MPN/g of feces; p < 0.01). Cattle performance was not affected by SRP administration. CONCLUSIONS The E. coli O157:H7 SRP-based vaccine might serve as a preharvest intervention to reduce the burden of E. coli O157:H7 on cattle presented for harvest.

Escherichia coli O157:H7 strains that persist in feedlot cattle are genetically related and demonstrate an enhanced ability to adhere to intestinal epithelial cells.

ABSTRACT A longitudinal study was conducted to investigate the nature of Escherichia coli O157:H7 colonization of feedlot cattle over the final 100 to 110 days of finishing. Rectal fecal grab samples were collected from an initial sample population of 788 steers every 20 to 22 days and microbiologically analyzed to detect E. coli O157:H7. The identities of presumptive colonies were confirmed using a multiplex PCR assay that screened for gene fragments unique to E. coli O157:H7 (rfbE and fliCh7) and other key virulence genes (eae, stx1, and stx2). Animals were classified as having persistent shedding (PS), transient shedding (TS), or nonshedding (NS) status if they consecutively shed the same E. coli O157:H7 genotype (based on the multiplex PCR profile), exhibited variable E. coli O157 shedding, or never shed morphologically typical E. coli O157, respectively. Overall, 1.0% and 1.4% of steers were classified as PS and NS animals, respectively. Characterization of 132 E. coli O157:H7 isolates from PS and TS animals by pulsed-field gel electrophoresis (PFGE) typing yielded 32 unique PFGE types. One predominant PFGE type accounted for 53% of all isolates characterized and persisted in cattle throughout the study. Isolates belonging to this predominant and persistent PFGE type demonstrated an enhanced (P < 0.0001) ability to adhere to Caco-2 human intestinal epithelial cells compared to isolates belonging to less common PFGE types but exhibited equal virulence expression. Interestingly, the attachment efficacy decreased as the genetic divergence from the predominant and persistent subtype increased. Our data support the hypothesis that certain E. coli O157:H7 strains persist in feedlot cattle, which may be partially explained by an enhanced ability to colonize the intestinal epithelium.

Validation of the use of organic acids and acidified sodium chlorite to reduce Escherichia coli O157 and Salmonella typhimurium in beef trim and ground beef in a simulated processing environment

A study was conducted to determine if acidified sodium chlorite (1,200 ppm) and acetic and lactic acids (2 and 4%) were effective in reducing foodborne pathogens in beef trim prior to grinding in a simulated processing environment. The reduction of Salmonella Typhimurium and Escherichia coli O157:H7 at high (4.0 log CFU/g) and low (1.0 log CFU/g) inoculation doses was evaluated at various processing steps, including the following: (i) in trim just after treatment application, (ii) in ground beef just after grinding, (iii) in ground beef 24 h after refrigerated storage, (iv) in ground beef 5 days after refrigerated storage, and (v) in ground beef 30 days after frozen storage. All antimicrobial treatments reduced the pathogens on the trim inoculated with the lower inoculation dose to nondetectable numbers in the trim and in the ground beef. There were significant reductions of both pathogens in the trim and in the ground beef inoculated with the high inoculation doses. On the trim itself, E. coli O157:H7 and Salmonella Typhimurium were reduced by 1.5 to 2.0 log cycles, with no differences among all treatments. In the ground beef, the organic acids were more effective in reducing both pathogens than the acidified sodium chlorite immediately after grinding, but after 1 day of storage, there were no differences among treatments. Overall, in the ground beef, there was a 2.5-log reduction of E. coli O157:H7 and a 1.5-log reduction of Salmonella Typhimurium that was sustained over time in refrigerated and frozen storage. Very few sensory differences between the control samples and the treated samples were detected by a consumer panel. Thus, antimicrobial treatments did not cause serious adverse sensory changes. Use of these antimicrobial treatments can be a promising intervention available to ground beef processors who currently have few interventions in their process.

WGS accurately predicts antimicrobial resistance in Escherichia coli

OBJECTIVES The objective of this study was to determine the effectiveness of WGS in identifying resistance genotypes of MDR Escherichia coli and whether these correlate with observed phenotypes. METHODS Seventy-six E. coli strains were isolated from farm cattle and measured for phenotypic resistance to 15 antimicrobials with the Sensititre(®) system. Isolates with resistance to at least four antimicrobials in three classes were selected for WGS using an Illumina MiSeq. Genotypic analysis was conducted with in-house Perl scripts using BLAST analysis to identify known genes and mutations associated with clinical resistance. RESULTS Over 30 resistance genes and a number of resistance mutations were identified among the E. coli isolates. Resistance genotypes correlated with 97.8% specificity and 99.6% sensitivity to the identified phenotypes. The majority of discordant results were attributable to the aminoglycoside streptomycin, whereas there was a perfect genotype-phenotype correlation for most antibiotic classes such as tetracyclines, quinolones and phenicols. WGS also revealed information about rare resistance mechanisms, such as structural mutations in chromosomal copies of ampC conferring third-generation cephalosporin resistance. CONCLUSIONS WGS can provide comprehensive resistance genotypes and is capable of accurately predicting resistance phenotypes, making it a valuable tool for surveillance. Moreover, the data presented here showing the ability to accurately predict resistance suggest that WGS may be used as a screening tool in selecting anti-infective therapy, especially as costs drop and methods improve.

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.

Salmonella enterica Burden in Harvest-Ready Cattle Populations from the Southern High Plains of the United States

ABSTRACT Our objectives were to quantify the Salmonella enterica burdens in harvest-ready cattle and to identify specific at-risk populations of cattle most likely to harbor multiply resistant S. enterica. Hide swabs were collected in abattoirs from three cohorts of cattle (feedlot origin cattle that had achieved desirable harvest characteristics and dairy- and beef-type cows harvested because of poor productivity). Feces were collected from two cohorts housed in feedlots (cattle that had achieved desirable harvest characteristics and animals identified for salvage recovery because of poor productivity). Facilities were visited on four occasions over a 12-month period. Salmonella enterica isolates were recovered, and organisms were quantified using standard microbiological methodologies. Susceptibility to antimicrobial drugs and serotype were determined for one S. enterica isolate per sample. Salmonella enterica was recovered from 55.6% of 1,681 samples. The prevalences on hides and in feces were 69.6% and 30.3%, respectively. The concentrations of S. enterica organisms averaged (as determined by the most probable number technique) 1.82 log10/100 cm2 of hides and 0.75 log10/g of feces. None of the isolates recovered from cattle that had achieved desirable harvest characteristics were resistant to four or more drugs. For isolates recovered from animals with poor productivity characteristics, 6.5% were resistant to four or more drugs. Twenty-two serovars were identified, with the most common being Salmonella enterica serovar Anatum (25.5%), Salmonella enterica serovar Montevideo (22.2%), and Salmonella enterica serovar Cerro (12.5%). High-level resistance, i.e., resistance to four or more drugs, was clustered within a few relatively uncommon serovars. These results demonstrate that even though S. enterica isolates are readily recoverable from harvest-ready cattle, multiply resistant variants are rare and are associated with specific serovars in cattle harvested because of poor productivity characteristics.