Reduction of Escherichia coli O157:H7 in Finishing Cattle Fed Corn Genetically Modified to Produce Increased Concentrations of Alpha Amylase in the Corn Kernel.

Cattle are recognized as the principal reservoir for Escherichia coli O157:H7 and preharvest food safety efforts often focus on decreasing shedding of this pathogen in cattle feces. Enogen® corn (EC; Syngenta Seeds, LLC) is genetically modified to produce enhanced concentrations of α-amylase in the corn kernel endosperm. Research has demonstrated improvements in feed efficiency for cattle fed EC and research has not yet explored whether improved digestion impacts foodborne pathogen populations in cattle. Therefore, this study explored effects of finishing diets containing EC on Escherichia coli O157:H7 prevalence in cattle. A 2 × 2 factorial experiment was conducted with steers (n = 960) fed diets consisting of 2 types of silage (EC or Control) and grain (EC or Control), fed daily ad libitum. Steers were grouped into 12 blocks by incoming body weight, blocks were randomly assigned to one of four pens, and pens were randomly assigned to one diet. Cattle were sampled using rectoanal mucosal swabs in cohorts of 298-337 cattle per day, for a total of 3 sampling days (15-16 days apart). Escherichia coli O157:H7 prevalence rates ranged from not detected (0/75) to 10.0% (8/80) depending on sampling day. Tests for the silage × corn interaction, and the main effects of silage and corn, were not significant (p > 0.05); however, EC reduced the odds of Escherichia coli O157:H7 prevalence by 43% compared to the control corn diet (p = 0.07). Diets containing EC tended to decrease Escherichia coli O157:H7 prevalence in feedlot cattle; however, this reduction was not significant. Before a conclusion can be drawn about impact of EC on Escherichia coli O157:H7 in cattle, further research is necessary to (1) determine if this tendency is due to increased alpha amylase activity and (2) elucidate impact on Escherichia coli O157:H7 prevalence and concentration, as well as a possible mechanism of action.

Impact of the Probiotic Organism Megasphaera elsdenii on Escherichia coli O157:H7 Prevalence in Finishing Cattle.

Feedlot cattle commonly shed the foodborne pathogen Escherichia coli O157:H7 in their feces. Megasphaera elsdenii (ME), a lactic acid-utilizing bacterium, is commonly administered to cattle to avoid lactate accumulation in the rumen and to control ruminal acidosis. The impact of administering ME on foodborne pathogen prevalence, specifically E. coli O157:H7, has not been explored. The purpose of this study was to quantify E. coli O157:H7 prevalence in finishing cattle administered ME. Cattle (n = 448) were assigned to treatments in a randomized complete block design with repeated measurements over two sampling periods. Treatments were arranged as a 2 × 2 factorial containing: ruminally protected lysine (RPL; included for a complementary study) fed at 0% or 0.45% of diet dry matter; with or without ME. Freeze-dried ME was administered as an oral drench (1 × 1010 CFU/steer on day one) and then top dressed onto basal diets (1 × 107 CFU/steer) daily thereafter. Rectoanal mucosal swabs (RAMS) were obtained from animals before harvest to determine the E. coli O157:H7 prevalence. The inclusion of RPL (P = 0.2136) and ME (P = 0.5012) did not impact E. coli O157:H7 prevalence, and RPL was not included in any significant interactions (P > 0.05). A significant interaction was observed between ME and sampling period (P = 0.0323), indicating that the effect of ME on E. coli O157:H7 prevalence varied over the sampling period. A diet containing ME reduced the odds of E. coli O157:H7 prevalence by 50% during sampling period 1 (8.0% and 14.7% for cattle with and without ME, respectively) and increased the odds by 23% during sampling period 2 (10.8% and 8.9% for cattle with and without ME, respectively). Administering ME in cattle diets did not impact E. coli O157:H7 in feedlot cattle. This is the first study to investigate the use of ME as a preharvest food safety intervention in cattle, and additional research is necessary to determine the efficacy.

Rapid Quantitative Method Development for Beef and Pork Lymph Nodes Using BAX® System Real Time Salmonella Assay.

The goal of this study was to develop a rapid RT-PCR enumeration method for Salmonella in pork and beef lymph nodes (LNs) utilizing BAX®-System-SalQuant® as well as to assess the performance of the methodology in comparison with existing ones. For study one: PCR curve development, pork, and beef LNs (n = 64) were trimmed, sterilized, pulverized, spiked with 0.00 to 5.00 Log CFU/LN using Salmonella Typhimurium, and then homogenized with BAX-MP media. Samples were incubated at 42 °C and tested at several time points using the BAX®-System-RT-PCR Assay for Salmonella. Cycle-Threshold values from the BAX®-System, for each Salmonella concentration were recorded and utilized for statistical analysis. For study two: Method comparison; additional pork and beef LNs (n = 52) were spiked and enumerated by (1) 3M™EB-Petrifilm™ + XLD-replica plate, (2) BAX®-System-SalQuant®, and (3) MPN. Linear-fit equations for LNs were estimated with recovery times of 6 h and a limit of quantification (LOQ) of 10 CFU/LN. Slopes and intercepts for LNs using BAX®-System-SalQuant® when compared with MPN were not significantly different (p < 0.05), while the same parameters for 3M™EB-Petrifilm™ + XLD-replica plate were significantly different (p > 0.05). The results support the capability of BAX®-System-SalQuant® to enumerate Salmonella in pork and beef LNs. This development adds support to the use of PCR-based quantification methodologies for pathogen loads in meat products.

The Use of Probiotic Megasphaera elsdenii as a Pre-Harvest Intervention to Reduce Salmonella in Finishing Beef Cattle: An In Vitro Model.

Reducing Salmonella in cattle may mitigate the risk of transmission through the food chain. Megasphaera elsdenii (ME) is a microorganism found naturally in the bovine rumen that can be administered as a probiotic to mitigate ruminal acidosis. Understanding the impact of feeding ME to Salmonella populations in cattle was the objective of this study. Bovine ruminal fluid (RF) and feces were inoculated with antibiotic susceptible or resistant Salmonella and treated with varying concentrations of ME. Salmonella was enumerated at 0, 24, 48, and 72 h using the most probable number (MPN). Volatile fatty acids (VFAs) and pH were recorded from non-inoculated samples. Treating RF with ME did not significantly impact Salmonella concentration or VFA production (p > 0.05). The pH of RF and feces decreased over time (p ≤ 0.05). Salmonella concentration declined in feces, with the largest reduction of 1.92 log MPN/g and 1.05 log MPN/g observed for antibiotic susceptible Salmonella between 0 and 72 h by the 2.5 × 105 CFU/g and control (0.0 CFU/g) concentration of ME, respectively. Treating RF with ME did not impact Salmonella concentration. Salmonella concentration in feces decreased, although ME must be further investigated before a conclusion regarding efficacy in vitro can be determined.

Attachment of Shiga Toxin-Producing Escherichia coli (STEC) to Pre-Chill and Post-Chill Beef Brisket Tissue.

Shiga toxin-producing Escherichia coli (STEC) has caused numerous foodborne illness outbreaks where beef was implicated as the contaminated food source. Understanding how STEC attach to beef surfaces may inform effective intervention applications at the abattoir. This simulated meat processing conditions to measure STEC attachment to adipose and lean beef tissue. Beef brisket samples were warmed to a surface temperature of 30 °C (warm carcass), while the remaining samples were maintained at 4 °C (cold carcass), prior to surface inoculation with an STEC cocktail (O26, O45, O103, O111, O121, O145, and O157:H7). Cocktails were grown in either tryptic soy broth (TSB) or M9 minimal nutrient medium. Loosely and firmly attached cells were measured at 0, 3, 5, and 20 min and 1, 3, 8, 12, 24 and 48 h. TSB-grown STEC cells became more firmly attached throughout storage and a difference in loosely versus firmly attached populations on lean and adipose tissues was observed. M9-grown STEC demonstrated a 0.2 log10 CFU/cm2 difference in attachment to lean versus adipose tissue and variability in populations was recorded throughout sampling. Future research should investigate whether a decrease in intervention efficacy correlates to an increase in firmly attached STEC cells on chilled carcasses and/or subprimals, which has been reported.

Validation of post-harvest antimicrobial interventions to control Shiga toxin-producing Escherichia coli (STEC) on market hog carcass surfaces.

Although swine are less associated with STEC foodborne disease outbreaks, the potential for swine to serve as a source of STEC infections in human beings cannot be disregarded. This study compared eight USDA-approved antimicrobial intervention technologies to quantify their ability to reduce STEC contamination on market hog carcasses. Hogs were harvested to provide skin-on carcass sides, and eight sides (per three replications) were inoculated with a 7-strain STEC cocktail (ca. 5 log CFU/cm2 across all external and body cavity surfaces). Each side was randomly assigned to a final pre-chill wash treatment administered in a commercial Chad carcass cabinet using a low-volume spray [3% lactic acid (lLA; 130 °F), 400 ppm peracetic acid (lPAA), or acidified 400 ppm peracetic acid (laPAA)] or a high-volume wash [ambient water (hAW), 400 ppm PAA (hPAA), 400 or 600 ppm hypobromous acid (hDBDMH), or 71 °C water (hHW)] treatment according to a randomized complete block study design. Post-treatment (after a 10-min hanging drip) and post-chilling (18 h at 2 °C) STEC reductions were compared for external skin-on surfaces and internal body cavity lean surface tissue. Post-treatment color changes were determined for lean, adipose, and skin carcass surfaces before and after chilling. When applied to the external, skin-on surface, the hHW, hPAA, and hDBDMH600 deluge washes were significantly (P ≤ 0.05) more effective than the other intervention technologies, achieving STEC reductions of 3.8, 3.4, and 3.2 log CFU/cm2, respectively. In comparison, the hAW control reduced STEC by 1.7-log CFU/cm2 on the external, skin-on surface. The carcass interventions were less effective at reducing STEC populations attached to interior body cavity (diaphragm region), with post-chill populations reduced by 0.9-2.2 log cycles, while the hAW control wash achieved a 0.6-log reduction. None of the treatments negatively impacted instrumental carcass color. While all market hog carcass interventions reduced STEC populations, larger reductions were observed when applied to the external, skin-on surface, with the largest reductions achieved by the hHW, hPAA, and hDBDMH600 deluge washes. These data equip pork processors with the information necessary to support decision-making when selecting an intervention technology.

Investigation into the Efficacy of Bdellovibrio bacteriovorus as a Novel Preharvest Intervention To Control Escherichia coli O157:H7 and Salmonella in Cattle Using an In Vitro Model.

Cattle are an important reservoir for the foodborne pathogens Salmonella and Escherichia coli O157:H7; they frequently harbor these microorganisms in their digestive tracts and shed them in their feces. Thus, there is potential for contamination of cattle hides and, subsequently, carcasses. Interventions aimed at reducing or eliminating pathogen shedding preharvest will also reduce the likelihood of beef product contamination by these pathogens. Therefore, this study used an in vitro model to evaluate Bdellovibrio bacteriovorus, a gram-negative microorganism that preys upon other gram-negative microorganisms, as a preharvest intervention to control Salmonella and E. coli O157:H7. Rumen fluid and feces were inoculated with pansusceptible or antimicrobial-resistant strains of one pathogen. Control samples were treated with HEPES buffer, whereas experimental samples were exposed to HEPES buffer plus B. bacteriovorus. Salmonella and E. coli O157:H7 populations were quantified at 0, 24, 48, and 72 h. The most-probable-number (MPN) technique, followed by streaking onto xylose lysine Tergitol 4 agar, was used to determine Salmonella populations, whereas spread plating onto sorbitol MacConkey agar supplemented with cefixime and tellurite was employed to enumerate E. coli O157:H7. B. bacteriovorus reduced pansusceptible Salmonella in cattle feces by 2.02 Log MPN/g (P = 0.0005) and antimicrobial-resistant Salmonella by 3.79 (P < 0.0001) and 2.24 (P = 0.0013) Log MPN/g after 24 and 48 h, respectively, in comparison to control samples. Significant reductions were not observed for E. coli O157:H7 in rumen or feces. These data suggest that further investigation into B. bacteriovorus efficacy as a preharvest intervention to control Salmonella in cattle is warranted.

Salmonella in beef and produce from honduras.

Salmonella continues to cause a considerable number of foodborne illnesses worldwide. The sources of outbreaks include contaminated meat and produce. The purpose of this study was to establish an initial investigation of the burden of Salmonella in produce and beef from Honduras by sampling retail markets and abattoirs. Retail produce samples (cantaloupes, cilantro, cucumbers, leafy greens, peppers, and tomatoes; n = 573) were purchased in three major cities of Honduras, and retail whole-muscle beef (n = 555) samples were also purchased in four major cities. Additionally, both hide and beef carcass (n = 141) samples were collected from two Honduran abattoirs. Whole-muscle beef samples were obtained using a sponge hydrated with buffered peptone water, and 10 ml of the buffered peptone water rinsate of each produce sample was collected with a dry sponge and placed in a bag to be transported back to the United States. Salmonella was detected using a commercially available, closeplatform PCR system, and positive samples were subjected to culture on selective media to obtain isolates. Overall, the prevalence of Salmonella-positive samples, based on PCR detection in Honduras (n = 555) retail beef was 10.1% (95% confidence interval = 7.8, 12.9), whereas 7.8% (n = 141) of beef carcass and hides samples were positive in both beef plants. The overall Salmonella prevalence for all produce samples (n = 573) collected was 2.1% (95% confidence interval = 1.2, 3.6). The most common serotypes identified in Honduras were Salmonella Typhimurium followed by Derby. These results provide an indication of Salmonella contamination of beef and produce in Honduras. Developing a Salmonella baseline for Latin America through an initial investigation like the one presented here contributes to a broader global understanding of the potential exposure through food, thus providing insight into the needs for control strategies.

Substantial within-animal diversity of Salmonella isolates from lymph nodes, feces, and hides of cattle at slaughter.

Lymph nodes (mandibular, mesenteric, mediastinal, and subiliac; n = 68) and fecal (n = 68) and hide (n = 35) samples were collected from beef carcasses harvested in an abattoir in Mexico. Samples were analyzed for Salmonella, and presumptive colonies were subjected to latex agglutination. Of the isolates recovered, a subset of 91 was characterized by serotyping, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility phenotyping. Salmonella was isolated from 100% (hide), 94.1% (feces), 91.2% (mesenteric), 76.5% (subiliac), 55.9% (mandibular), and 7.4% (mediastinal) of samples. From the 87 typeable isolates, eight Salmonella enterica serotypes, including Kentucky (32.2%), Anatum (29.9%), Reading (17.2%), Meleagridis (12.6%), Cerro (4.6%), Muenster (1.1%), Give (1.1%), and Mbandaka (1.1%), were identified. S. Meleagridis was more likely (P = 0.03) to be recovered from lymph nodes than from feces or hides, whereas S. Kentucky was more likely (P = 0.02) to be recovered from feces and hides than from lymph nodes. The majority (59.3%) of the Salmonella isolates were pansusceptible; however, multidrug resistance was observed in 13.2% of isolates. Typing by PFGE revealed that Salmonella strains generally clustered by serotype, but some serotypes (Anatum, Kentucky, Meleagridis, and Reading) were comprised of multiple PFGE subtypes. Indistinguishable PFGE subtypes and, therefore, serotypes were isolated from multiple sample types, and multiple PFGE subtypes were commonly observed within an animal. Given the overrepresentation of some serotypes within lymph nodes, we hypothesize that certain Salmonella strains may be better at entering the bovine host than other Salmonella strains or that some may be better adapted for survival within lymph nodes. Our data provide insight into the ecology of Salmonella within cohorts of cattle and offer direction for intervention opportunities.

Cross-sectional study examining Salmonella enterica carriage in subiliac lymph nodes of cull and feedlot cattle at harvest.

Bovine peripheral lymph nodes (LNs), including subiliac LNs, have been identified as a potential source of human exposure to Salmonella enterica, when adipose trim containing these nodes is incorporated into ground beef. In order to gain a better understanding of the burden of S. enterica in peripheral LNs of feedlot and cull cattle, a cross-sectional study was undertaken in which 3327 subiliac LNs were collected from cattle at harvest in seven plants, located in three geographically distinct regions of the United States. Samples were collected in three seasons: Fall 2010, Winter/Spring 2011, and Summer/Fall 2011. A convenience sample of 76 LNs per day, 2 days per season (approximately 1 month apart), was collected per plant, from carcasses held in the cooler for no less than 24 h. Every 10(th) carcass half on a rail was sampled, in an attempt to avoid oversampling any single cohort of cattle. Median point estimates of S. enterica contamination were generally low (1.3%); however, median Salmonella prevalence was found to be greater in subiliac LNs of feedlot cattle (11.8%) compared to those of cull cattle (0.65%). Enumeration analysis of a subset of 618 feedlot cattle LNs showed that 67% of those harboring S. enterica (97 of 144) did so at concentrations ranging from <0.1 to 1.8 log10 CFU/g, while 33% carried a higher burden of S. enterica, with levels ranging from 1.9 to >3.8 log10 CFU/g. Serotyping of S. enterica isolated identified 24 serotypes, with the majority being Montevideo (44.0%) and Anatum (24.8%). Antimicrobial susceptibility phenotypes were determined for all isolates, and the majority (86.1%) were pansusceptible; however, multidrug-resistant isolates (8.3%) were also occasionally observed. As Salmonella contained within LNs are protected from carcass interventions, research is needed to define opportunities for mitigating the risk of Salmonella contamination in LNs of apparently healthy cattle.

Implementation of targeted interventions to control Escherichia coli O157:H7 in a commercial abattoir.

The objective of this study was to define locations on the carcass with highest contamination of E. coli O157 throughout the harvest process and implement targeted interventions to reduce or eliminate contamination. To establish a pathogen baseline, samples were collected at the foreshank, hindshank, inside round, neck and midline area and evaluated for E. coli O157:H7 presence. Environmental samples were also collected in the harvest area and the fabrication area of the facility. E. coli O157:H7 prevalence was highest on the foreshank, hindshank and inside rounds in the baseline study and steam vacuums/cones were implemented as an intervention in these specific areas on the harvest floor. At pre-evisceration, foreshank prevalence of E. coli O157:H7 was significantly (P<0.05) reduced from 21.7% to 3.1% after the application of steam interventions. At the final rail, foreshank prevalence in the baseline study was 4.2% while no E. coli O157:H7 was detected post-intervention implementation. E. coli O157:H7 on hindshanks and inside rounds was significantly reduced after intervention implementation from 24.2 to 11.5% and 37.5 to 16.7%, respectively at the final rail. Pathogen contamination of environmental samples collected in fabrication declined from 6.7% to 0.7% after slaughter interventions were implemented. Data indicate the identifying areas of contamination on the carcass and implementing interventions can significantly reduce E. coli O157 on the carcasses and in the fabrication environment.