Effect of a Saccharomyces cerevisiae Postbiotic Feed Additive on Salmonella Enteritidis Colonization of Cecal and Ovarian Tissues in Directly Challenged and Horizontally Exposed Layer Pullets.

Determining the efficacy of feed-additive technologies utilized as pre-harvest food-safety interventions against Salmonella enterica may be influenced by factors including, but not limited to, mechanism of action, experimental design variables, Salmonella serovar(s), exposure dose, route, or duration in both controlled research and real-world field observations. The purpose of this study was to evaluate the dietary inclusion of a Saccharomyces cerevisiae fermentation-derived postbiotic (SCFP) additive (Diamond V, Original XPC®) on the colonization of cecal and ovarian tissues of commercial pullets directly and indirectly exposed to Salmonella Enteritidis (SE). Four hundred and eighty commercial, day-of-age W-36 chicks were randomly allotted to 60 cages per treatment in two identical BSL-2 isolation rooms (Iowa State University) with four birds per cage and fed control (CON) or treatment (TRT) diets for the duration of study. At 16 weeks, two birds per cage were directly challenged via oral gavage with 1.1 × 109 CFU of a nalidixic-acid-resistant SE strain. The remaining two birds in each cage were thus horizontally exposed to the SE challenge. At 3, 7, and 14 days post-challenge (DPC), 20 cages per group were harvested and sampled for SE prevalence and load. No significant differences were observed between groups for SE prevalence in the ceca or ovary tissues of directly challenged birds. For the indirectly exposed cohort, SE cecal prevalence at 7 DPC was significantly lower for TRT (50.0%) vs. CON (72.5%) (p = 0.037) and, likewise, demonstrated significantly lower mean SE cecal load (1.69 Log10) vs. CON (2.83 Log10) (p = 0.005). At 14 DPC, no significant differences were detected but ~10% fewer birds remained positive in the TRT group vs. CON (p > 0.05). These findings suggest that diets supplemented with SCFP postbiotic may be a useful tool for mitigating SE colonization in horizontally exposed pullets and may support pre-harvest food-safety strategies.

Dietary Inclusion of a Saccharomyces cerevisiae-Derived Postbiotic Is Associated with Lower Salmonella enterica Burden in Broiler Chickens on a Commercial Farm in Honduras.

Postbiotic feed additives may aid foodborne pathogen reduction during poultry rearing. The study objective was to evaluate a postbiotic additive in parallel to an industry control diet and the subsequent associated burden of Salmonella enterica on a single, commercial broiler farm in Honduras. Twelve houses were matched and assigned the standard diet (CON) or standard diet plus postbiotic (SCFP). New litter was placed in each house and retained across flock cycles with sampling prior to each chick placement and three consecutive rearing cycles. At ~33-34 days, 25 ceca were collected on-farm from each house, treatment, and cycle. Salmonella prevalence in litter for CON (30.6%) and SCFP (27.8%) were equivalent; however, Salmonella load within positive samples was lower (p = 0.04) for SCFP (3.81 log10 MPN/swab) compared to CON (5.53 log10 MPN/swab). Cecal prevalence of Salmonella was lower (p = 0.0006) in broilers fed SCFP (3.4%) compared to CON (12.2%). Salmonella load within positive ceca were numerically reduced (p = 0.121) by 1.45 log10 MPN/g for SCFP (2.41 log10 MPN/g) over CON (3.86 log10 MPN/g). Estimated burden was lower (p = 0.003) for SCFP flocks (3.80 log10 MPN) compared to CON (7.31 log10 MPN). These data demonstrate the preharvest intervention potential of postbiotics to reduce Salmonella enterica in broiler chickens.

Application of a Commercial Salmonella Real-Time PCR Assay for the Detection and Quantitation of Salmonella enterica in Poultry Ceca.

ABSTRACT: Foodborne salmonellosis is commonly associated with poultry and poultry products, necessitating continued development of pre- and postharvest food safety interventions and risk management strategies. Evaluation of technologies and strategies is limited by availability of cost-effective, rapid laboratory methods. The objective of this study was to evaluate a commercial qualitative PCR assay and its novel quantitative application to detect and enumerate Salmonella in poultry ceca as an analytical matrix. Ceca were collected at harvest, the contents were homogenized, and paired samples were evaluated with buffered peptone water (BPW) and BAX MP + Supplement (MPS) preenrichment broths followed by PCR screening with a BAX System Q7 PCR and by culture isolation. Additional ceca were inoculated with Salmonella to develop a standard curve for the BAX System SalQuant quantitative PCR application (QA), and estimates were obtained by the QA and most-probable-number (MPN) methods. For preenrichment media, PCR outcomes were equivalent to those of culture isolation for detecting Salmonella in ceca with 95.65 and 87.88% sensitivity and 82.00 and 100.00% specificity (P = 0.074) for BPW and MPS, respectively. However, at the sample level, BPW performed significantly worse (47.92%) than did MPS (68.75%) for overall isolation of Salmonella (P < 0.0001). After standard curve development, the mean QA estimates obtained for the inoculated samples were 1.14 (95% confidence interval [CI]: 0.62 to 1.66), 1.79 (1.50 to 2.08), 2.91 (2.65 to 3.17), and 3.76 (3.26 to 4.25) log CFU/mL for each targeted inoculation of 1.0, 2.0, 3.0, and 4.0 log CFU/mL, respectively, and were within or comparable to the 95% CI values of paired MPN estimates. These data support the use of MPS for the detection and isolation of Salmonella enterica from poultry ceca when screening with PCR and indicate that QA may be useful as an alternative tool to estimate Salmonella loads in poultry ceca, which may support preharvest food safety interventions.

Determination of Antimicrobial Resistance Patterns in Salmonella from Commercial Poultry as Influenced by Microbiological Culture and Antimicrobial Susceptibility Testing Methods.

Monitoring antimicrobial resistance of foodborne pathogens in poultry is critical for food safety. We aimed to compare antimicrobial resistance phenotypes in Salmonella isolated from poultry samples as influenced by isolation and antimicrobial susceptibility testing methods. Salmonella isolates were cultured from a convenience sample of commercial broiler ceca with and without selective broth enrichment, and resistance phenotypes were determined for 14 antimicrobials using the Sensititre® platform and a qualitative broth breakpoint assay. The broth breakpoint method reported higher resistance to chloramphenicol, sulfisoxazole, and the combination of trimethoprim and sulfamethoxazole, and lower resistance to streptomycin as compared to the Sensititre® assay in trial one. Selective enrichment of samples containing Salmonella in Rappaport-Vassiliadis broth reported lowered detectable resistance to amoxicillin/clavulanic acid, ampicillin, azithromycin, cefoxitin, ceftriaxone, nalidixic acid, and meropenem, and increased resistance to streptomycin and tetracycline than direct-plating samples in trial one. Using matched isolates in trial two, the Sensititre® assay reported higher resistance to chloramphenicol and gentamicin, and lower resistance to nalidixic acid as compared to the broth breakpoint method. These results suggest methodology is a critical consideration in the detection and surveillance of antimicrobial resistance phenotypes in Salmonella isolates from poultry samples and could affect the accuracy of population or industry surveillance insights and intervention strategies.

Corn-Based Distillers' Grains in Diets for Feedlot Cattle Are Associated with the Burden of Escherichia coli O157 in Feces.

Inclusion of distillers' grains (DGs) has been associated with increased prevalence of Escherichia coli O157:H7 in cattle housed in research settings. Our objective was to quantify the relationship between inclusion of DGs in commercial feedlot rations and the burden of E. coli O157. A convenience sample of 10 feedlots was enrolled based on DG use in finishing diets; 1 cohort included 5 feedlots in which DGs were greater than 15% of the dietary dry matter and the other cohort consisted of 5 feedlots at a concentration less than 8%. Sampling occurred at each feedlot on four occasions at ∼6-week intervals. At each feedlot visit, 4 pens of cattle within 3 weeks of slaughter were selected and 24 freshly voided fecal pats were sampled. Ten-gram samples were enriched in 90 mL of modified tryptic soy broth with novobiocin (20 mg/L) for 14 h at 42°C. Enrichments were subjected to immunomagnetic separation, plating onto chromogenic agar with novobiocin (5 mg/L) and potassium tellurite (2.5 mg/L), incubation for 18 h at 37°C, and latex agglutination of morphologically typical colonies. E. coli O157 was recovered from 16.7% of 3840 samples. Adjusted prevalence was 14.3% after controlling for within-feedlot and within-pen clustering. Prevalence during each sampling period was 19.9% (round 1), 21.0% (round 2), 14.1% (round 3), and 11.7% (round 4). Prevalence varied between cohorts, but this difference varied over time (p = 0.06). Among those with greater than 15% of the diet as DGs, prevalence was greater than those with less than 8% inclusion for all rounds of sampling (p < 0.01). Averaged across time, prevalence was 23.9% and 9.4% for those with greater than 15% and those with less than 8% of DGs, respectively. While observational, these data provide real-world support of reports of increased E. coli O157:H7 burden associated with DG use in cattle diets.

Method Modification for the Atlas Listeria Environmental LE Detection Assay Using FoodChek Actero Listeria Enrichment Media and Half-Fraser Media for the Detection of Listeria spp. from Environmental Surfaces.

Two candidate method modifications for the Atlas Listeria Environmental LE Detection Assay were compared with the U.S. Department of Agriculture (USDA)-Food Safety and Inspection Service Microbiology Laboratory Guidebook 8.09 (MLG 8.09) method for detection of Listeria spp. on stainless steel, polyvinyl chloride (PVC), and sealed concrete surfaces. For LE candidate method 1, samples were enriched in FoodChek Actero Listeria Enrichment Media [ALEM; Performance Tested MethodSM (PTM) 111201] at 35 ± 2°C for 18 to 24 h and evaluated for a range of analytical sample volumes. For LE candidate method 2, the current Roka PTM using 90 mL of Half-Fraser broth for enrichment at 35 ± 2°C was evaluated at 24 h with a reduced sample volume. These comparisons were made in multiple studies across the three environmental surfaces. Within each method and study, a total of 5 samples were uninoculated, 20 samples were inoculated with Listeria spp. at a low level to target fractional positivity, and 5 samples were inoculated with Listeria spp. at a high level to approach a probability of detection of 1. Inclusivity and exclusivity studies were also conducted for the LE method in combination with Half-Fraser and ALEM. The Atlas Listeria Environmental LE Detection Assay detected all 50 inclusive organisms, including 25 strains of L. monocytogenes and 5 strains of each of the other five common species of Listeria (L. innocua, L. welshimeri, L. ivanovii, L. seeligeri, and L. grayi) and none of the 30 exclusive organisms across all media and with both 200 and 2000 µL sample volumes. For the LE candidate method 1 studies, no significant differences were observed within the Roka ALEM method at 18, 20, or 24 h and for both the 200 and 2000 µL sample volumes as compared with the paired culture outcome. However, the ALEM method performed significantly better as compared with the unpaired reference method for sealed concrete and stainless steel. For the LE candidate method 2 studies, no significant differences were observed within the Roka HF method at 24 h for the 200 and 2000 µL samples as compared with the paired culture outcomes and unpaired reference method outcomes across the surfaces. The independent laboratory studies observed no significant differences in performance between the USDA/MLG 8.09 reference method and candidate methods 1 or 2, respectively, across the evaluated parameters. Overall, the candidate method 1 modification parameters and candidate method 2 sample parameters for the Atlas Listeria Environmental LE Detection Assay were statistically equivalent to or better than the reference method for detection of Listeria spp. on stainless steel, PVC, and sealed concrete surfaces, providing greater flexibility in method application for end users.

Developments in Rapid Detection Methods for the Detection of Foodborne Campylobacter in the United States.

The accurate and rapid detection of Campylobacter spp. is critical for optimal surveillance throughout poultry processing in the United States. The further development of highly specific and sensitive assays to detect Campylobacter in poultry matrices has tremendous utility and potential for aiding the reduction of foodborne illness. The introduction and development of molecular methods such as polymerase chain reaction (PCR) have enhanced the diagnostic capabilities of the food industry to identify the presence of foodborne pathogens throughout poultry production. Further innovations in various methodologies, such as immune-based typing and detection as well as high throughput analyses, will provide important epidemiological data such as the identification of unique or region-specific Campylobacter. Comparable to traditional microbiology and enrichment techniques, molecular techniques/methods have the potential to have improved sensitivity and specificity, as well as speed of data acquisition. This review will focus on the development and application of rapid molecular methods for identifying and quantifying Campylobacter in U.S. poultry and the emergence of novel methods that are faster and more precise than traditional microbiological techniques.

Rapid Detection and Classification of Salmonella enterica Shedding in Feedlot Cattle Utilizing the Roka Bioscience Atlas Salmonella Detection Assay for the Analysis of Rectoanal Mucosal Swabs.

With an increasing focus on preharvest food safety, rapid methods are required for the detection and quantification of foodborne pathogens such as Salmonella enterica in beef cattle. We validated the Atlas Salmonella Detection Assay (SEN), a nucleic acid amplification technology that targets Salmonella rRNA, for the qualitative detection of S. enterica with sample enrichment using immunomagnetic separation as a reference test, and we further evaluated its accuracy to predict pathogen load using SEN signal-to-cutoff (SCO) values from unenriched samples to classify animals as high or nonhigh shedders. Rectoanal mucosal swabs (RAMS) were collected from 238 beef cattle from five cohorts located in the Midwest or southern High Plains of the United States between July 2015 and April 2016. Unenriched RAMS samples were used for the enumeration and SEN SCO analyses. Enriched samples were tested using SEN and immunomagnetic separation methods for the detection of Salmonella. The SEN method was 100% sensitive and specific for the detection of Salmonella from the enriched RAMS samples. A SEN SCO value of 8, with a sensitivity of 93.5% and specificity of 94.3%, was found to be an optimum cutoff value for classifying animals as high or nonhigh shedders from the unenriched RAMS samples. The SEN assay is a rapid and reliable method for the qualitative detection and categorization of the shedding load of Salmonella from RAMS in feedlot cattle.

Salmonella and Campylobacter baseline in retail ground beef and whole-muscle cuts purchased during 2010 in the United States.

Salmonella enterica and Campylobacter spp. cause a considerable number of human illnesses each year, and the vast majority of cases are foodborne. The purpose of this study was to establish the baseline of Salmonella and Campylobacter in beef products purchased from U.S. retail markets. Sampling was carried out in 38 American cities. Retail raw ground and whole-muscle beef (n = 2,885) samples were purchased and examined for the presence of Salmonella. Samples testing positive for Salmonella were identified with the commercial BAX System, which is a real-time PCR-based system. Of the original samples purchased, 1,185 were selected and tested for the presence of Campylobacter. Positive samples were isolated via direct plating and confirmed via agglutination and biochemical testing. Salmonella was detected in 0.66% of the total samples purchased. The prevalence of Salmonella in ground beef packages was 0.42% for modified atmosphere packaging, 0.63% for chub packaging, and 0.59% for overwrapped packages. Salmonella was detected in 1.02% of whole-muscle cuts. There was no relationship (P = 0.18) between product type (ground or whole muscle) and the percentage of positive samples. Campylobacter was recovered from 9.3% of samples. A greater percentage (17.24%, P < 0.01) of whole-muscle cuts tested positive for Campylobacter compared with ground beef samples (7.35%). Estimating pathogen baselines in U.S. retail beef is essential for allotting resources and directing interventions for pathogen control. These data can be utilized for a more complete understanding of these pathogens and their impact on public health from the consumption of beef products.