Publications by authors named "Michael J Rothrock"

40 Publications

Mapping foodborne pathogen contamination throughout the conventional and alternative poultry supply chains.

Poult Sci 2021 Mar 27;100(7):101157. Epub 2021 Mar 27.

Department of Food Science and Technology, University of Georgia, 100 Cedar St., Athens, GA, USA. Electronic address:

Recently, there has been a consumer push for natural and organic food products. This has caused alternative poultry production, such as organic, pasture, and free-range systems, to grow in popularity. Due to the stricter rearing practices of alternative poultry production systems, different types of levels of microbiological risks might be present for these systems when compared to conventional production systems. Both conventional and alternative production systems have complex supply chains that present many different opportunities for flocks of birds or poultry meat to be contaminated with foodborne pathogens. As such, it is important to understand the risks involved during each step of production. The purpose of this review is to detail the potential routes of foodborne pathogen transmission throughout the conventional and alternative supply chains, with a special emphasis on the differences in risk between the two management systems, and to identify gaps in knowledge that could assist, if addressed, in poultry risk-based decision making.
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http://dx.doi.org/10.1016/j.psj.2021.101157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8182426PMC
March 2021

Using farm management practices to predict Campylobacter prevalence in pastured poultry farms.

Poult Sci 2021 Jun 11;100(6):101122. Epub 2021 Mar 11.

Department of Food Science and Technology, University of Georgia, Athens, GA, USA. Electronic address:

Contamination of poultry products by Campylobacter is often associated with farm management practices and processing plant practices. A longitudinal study was conducted on 11 pastured poultry farms in southeastern United States from 2014 to 2017. In this study, farm practices and processing variables were used as predictors for a random forest (RF) model to predict Campylobacter prevalence in pastured poultry farms and processing environments. Individual RF models were constructed for fecal, soil and whole carcass rinse after processing (WCR-P) samples. The performance of models was evaluated by the area under curve (AUC) from the receiver operating characteristics curve. The AUC values were 0.902, 0.894, and 0.864 for fecal, soil, and WCR-P models, respectively. Relative importance plots were generated to predict the most important variable in each RF model. Animal source of feces was identified as the most important variable in fecal model and the soy content of the brood feed was the most important variable for soil model. For WCR-P model, the average flock age showed the strongest impact on RF model. These RF models can help pastured poultry growers with food safety control strategies to reduce Campylobacter prevalence in pastured poultry farms.
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http://dx.doi.org/10.1016/j.psj.2021.101122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131732PMC
June 2021

The Effects of Feeding Antibiotic on the Intestinal Microbiota of Weanling Pigs.

Front Vet Sci 2021 12;8:601394. Epub 2021 Mar 12.

Department of Animal and Dairy Science, University of Georgia, Athens, GA, United States.

This study investigated the use of carbadox in the diet of nursery pigs. Ten pens of weanling piglets were assigned to 2 treatments: one containing carbadox and another without it. From days 21 to 35 of age, the first group of piglets was fed carbadox at 55 mg/kg of diet; followed by 27.5 mg/kg from days 36 to 49; and 0 mg/kg from days 50 to 63. The second group of pigs was fed a control diet without carbadox from days 21 to 63 of age. On days 35, 49, and 63, fecal samples were collected directly from the rectum of 2 piglets in each pen, and the samples were subjected to microbial DNA sequencing and metagenomic functional analysis using the 16S rRNA gene. Feed conversion from days 21 to 63 was improved ( = 0.04) in the group of piglets fed carbadox. Faith's phylogenetic diversity was similar ( = 0.89) for both groups of piglets on day 35, but it was diminished ( = 0.01) in the carbadox-fed group on day 49; however, following the complete removal of carbadox from their diets, this microbial diversity index was once again found to be similar ( = 0.27) in both groups on day 63. Likewise, abundances of , and were all similar between the two groups ( ≥ 0.40) on day 35, but were smaller in the carbadox group ( ≤ 0.05) on day 49; however, on day 63, abundances of all these genera were once again similar ( ≥ 0.29). Metabolic pathways involved in cellular growth, death, and genetic information processing (translation) were found to be similarly expressed in the microbiota of piglets from both groups on day 35 ( ≥ 0.52), but decreased in the carbadox group on day 49 ( ≤ 0.05), and were similar again in both groups on day 63 ( ≥ 0.51). These results revealed that feeding carbadox to piglets during the first 4 weeks after weaning significantly affected their fecal microbiotas; however, 2 weeks after the removal of carbadox, those changes tended to disappear, indicating that the shifts were carbadox-dependent.
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http://dx.doi.org/10.3389/fvets.2021.601394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996051PMC
March 2021

Dynamic Changes in the Gut Microbiome at the Acute Stage of Ischemic Stroke in a Pig Model.

Front Neurosci 2020 3;14:587986. Epub 2020 Dec 3.

Department of Foods and Nutrition, College of Family and Consumer Sciences, University of Georgia, Athens, GA, United States.

Stroke is a major cause of death and long-term disability affecting seven million adults in the United States each year. Recently, it has been demonstrated that neurological diseases, associated pathology, and susceptibility changes correlated with changes in the gut microbiota. However, changes in the microbial community in stroke has not been well characterized. The acute stage of stroke is a critical period for assessing injury severity, therapeutic intervention, and clinical prognosis. We investigated the changes in the gut microbiota composition and diversity using a middle cerebral artery (MCA) occlusion ischemic stroke pig model. Ischemic stroke was induced by cauterization of the MCA in pigs. Blood samples were collected prestroke and 4 h, 12 h, 1 day, and 5 days poststroke to evaluate circulating proinflammatory cytokines. Fecal samples were collected prestroke and 1, 3, and 5 days poststroke to assess gut microbiome changes. Results showed elevated systemic inflammation with increased plasma levels of tumor necrosis factor alpha at 4 h and interleukin-6 at 12 h poststroke, relative to prestroke. Microbial diversity and evenness were reduced at 1 day poststroke compared to prestroke. Microbial diversity at 3 days poststroke was negatively correlated with lesion volume. Moreover, beta-diversity analysis revealed trending overall differences over time, with the most significant changes in microbial patterns observed between prestroke and 3 days poststroke. Abundance of the Proteobacteria was significantly increased, while Firmicutes decreased at 3 days poststroke, compared to prestroke populations. Abundance of the lactic acid bacteria was reduced at 3 days poststroke. By day 5, the microbial pattern returned to similar values as prestroke, suggesting the plasticity of gut microbiome in an acute period of stroke in a pig model. These findings provide a basis for characterizing gut microbial changes during the acute stage of stroke, which can be used to assess stroke pathology and the potential development of therapeutic targets.
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http://dx.doi.org/10.3389/fnins.2020.587986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744295PMC
December 2020

The relationship between the rumen microbiome and carcass merit in Angus steers.

J Anim Sci 2020 Sep;98(9)

Department of Animal and Dairy Science, University of Georgia, Athens, GA.

The objective of this study was to explore the relationships between ruminal microbial populations from Angus steers that were divergent in carcass traits related to adipose accumulation. Twenty-four feedlot-finished Angus steers (age: 538 ± 21 d; body weight following lairage: 593.9 ± 43.7 kg) were slaughtered, and ruminal contents and carcass data were collected. Ruminal microbial deoxyribonucleic acid (DNA) extraction and 16S ribosomal ribonucleic acid (rRNA) gene sequencing were performed to determine microbial relative abundances, to estimate microbial diversity, and to predict microbial metabolic pathways. A variety of correlation analyses and one-way ANOVA were performed to investigate the relationships between the rumen microbiome and carcass traits. Marbling score (P = 0.001) and longissimus lipid content (P = 0.009) were positively correlated to Chao1 Richness Index, suggesting that increased intramuscular fat was associated with increased numbers of ruminal microbial species. The phyla Tenericutes and TM7 were negatively correlated (P ≤ 0.05) to marbling score and longissimus lipid content, indicating that lower abundances of these phyla may be associated with improvements in intramuscular fat content. Greater abundance of the bacterial family S24-7 was positively correlated (P = 0.002) to marbling score. Analysis by marbling classification revealed further linkages to microbial richness (P ≤ 0.063), diversity (P = 0.044), and S24-7 (P < 0.001) populations. Computational prediction of the microbial metabolic pathways revealed no differences (P ≥ 0.05) in metabolic pathway expression in rumen microbes between steers in the high- and low-marbling classes. Several phyla, families, and genera were positively correlated (P ≤ 0.05) to both rib fat thickness and yield grade. Collectively, our results suggest that microbial composition is associated to differing performance in carcass adipose traits. Overall, most of the bacterial taxa correlated to the intramuscular and subcutaneous fat depots did not overlap, suggesting the microbial population end products likely impacted adipose accumulation largely via separate adipogenic pathways of the host animal.
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http://dx.doi.org/10.1093/jas/skaa287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526868PMC
September 2020

The impact of feed efficiency selection on the ruminal, cecal, and fecal microbiomes of Angus steers from a commercial feedlot.

J Anim Sci 2020 Jul;98(7)

Department of Animal and Dairy Science, University of Georgia, Athens, GA.

Feed is the greatest cost of animal production, so reducing it is critical to increase producer profits. In ruminants, the microbial population within the gastrointestinal tract (GIT) is critical to nutrient digestion and absorption in both the rumen and the hindgut. The objective of this study was to determine the bacterial taxonomic profile of the rumen, cecum, and feces of feedlot steers at slaughter in order to link feed efficiency and the GIT bacterial populations from these three locations. Twenty commercial Angus steers were selected and divided into two groups according to their residual feed intake (RFI) classification determined during the feedlot-finishing period: high-RFI (n = 10) and low-RFI (n = 10). After the ruminal, cecal, and fecal samples were collected at slaughter, DNA extraction and 16S rRNA gene sequencing were performed on them to determine their bacterial composition. One-way ANOVA was performed on the animal performance data, alpha diversities, and bacterial abundances using RFI classification as the fixed effect. Overall, the ruminal bacterial population was the most different in terms of taxonomic profile compared with the cecal and fecal populations as revealed by beta diversity analysis (P < 0.001). Moreover, bacterial richness (Chao1) was greatest (P = 0.01) in the rumen of the high-RFI group compared with the low-RFI group. In contrast, bacterial richness and diversity in the intestinal environment showed that Chao1 was greater (P = 0.01) in the cecum, and the Shannon diversity index was greater in both the cecum and feces of low-RFI compared with high-RFI steers (P = 0.01 and P < 0.001, respectively). Ruminococcaceae was more abundant in the low-RFI group in the cecum and feces (P = 0.01); fecal Bifidobacteriaceae was more abundant in high-RFI steers (P = 0.03). No correlations (P ≥ 0.13) between any ruminal bacterial family and RFI were detected; however, Ruminococcaceae, Mogibacteriaceae, Christensenellaceae, and BS11 were negatively correlated with RFI (P < 0.05) in the cecum and feces. Succinivibrionaceae in the cecum was positively correlated with RFI (P = 0.05), and fecal Bifidobacteriaceae was positively correlated with RFI (P = 0.03). Results collectively indicate that in addition to the ruminal bacteria, the lower gut bacterial population has a significant impact on feed efficiency and nutrient utilization in feedlot steers; therefore, the intestinal bacteria should also be considered when examining the basis of ruminant feed efficiency.
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http://dx.doi.org/10.1093/jas/skaa230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392532PMC
July 2020

Assessment of a Potential Role of DSM 18020 as a Pectinase Producer for Utilization in Poultry Diets Based on Analyses.

Front Microbiol 2020 23;11:751. Epub 2020 Apr 23.

Department of Food Science and Center for Food Safety, University of Arkansas, Fayetteville, AR, United States.

Currently, the poultry industry has been faced with consumer pressure to utilize only vegetable feedstuffs in poultry diets, eliminate antibiotics from poultry production, and rear poultry in free range systems. To maintain current production standards, the industry must determine ways to enhance nutrient uptake and utilization further. One possible solution is the supplementation of pectinase, an enzyme that degrades pectin within the cell walls of plants, in poultry diets. Therefore, the objective of the current study was to determine the potential role of a pectinase producer, DSM 18020, as a commercially utilized pectinase producer in poultry diets against other known pectinase producers, . In the current study, whole genomes of DSM 18020 (Dd18020), 3937 (Dd3937), IPO 2222 (Ds2222), C-125 (BhC125), and subsp. str. 168 (Bs168) were compared using bioinformatic approaches to compare the chromosomal genome size, GC content, protein coding genes (CDS), total genes, average protein length (a.a.) and determine the predicted metabolic pathways, predicted pectin degrading enzymes, and pectin-degradation pathways across pectinase producers. Due to insufficient information surrounding the genome of Dd18020 (lack of annotation), the genome of Dd3937, a 99% identical genome to Dd18020, was utilized to compare pectinase-associated enzymes and pathways. The results from the current study demonstrated that Dd3937 possessed the most significant proportion of pathways presented and the highest number of pathways related to degradation, assimilation, and utilization of pectin. Also, Dd18020 exhibited a high number of pectinase-related enzymes. Both Dd3937 and Dd2222 shared the pectin degradation I pathway via the EC 3.1.1.11, EC 3.2.1.82, and EC 4.2.2.- enzymes, but did not share this pathway with either species. In conclusion, Dd18020 demonstrated the genetic potential to produce multiple pectinase enzymes that could be beneficial to the degradation of pectin in poultry diets. However, for Dd18020 to become a commercially viable enzyme producer for the poultry industry, further research quantifying the pectinase production and determining the stability of the produced pectinases during feed manufacturing are necessary.
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http://dx.doi.org/10.3389/fmicb.2020.00751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7191031PMC
April 2020

Gastrointestinal microbiomes of broilers and layer hens in alternative production systems.

Poult Sci 2020 Feb 24;99(2):660-669. Epub 2020 Jan 24.

Egg Safety & Quality Research Unit, U.S. National Poultry Research Center, USDA-ARS, Athens 30605, GA.

Alternative poultry production systems consisting of free-range or pasture flock raised poultry continues to increase in popularity. Based on the perceived benefits of poultry products generated from these alternative poultry production systems, they have commercial appeal to consumers. Several factors impact the health and well being of birds raised and maintained in these types of production systems. Exposure to foodborne pathogens and potential for colonization in the gastrointestinal tract has to be considered with these types of production systems. The gastrointestinal tract microbial composition and function of birds grown and maintained in alternative poultry operations may differ depending on diets, breed, and age of bird. Dietary variety and foraging behavior are potential influential factors on bird nutrition. The gastrointestinal tract microbiomes of birds raised under alternative poultry production systems are now being characterized with next-generation sequencing to identify individual microbial members and assess the impact of different factors on the diversity of microbial populations. In this review, the gastrointestinal tract microbiota contributions to free-range or pasture-raised broiler and egg layer production systems, subsequent applications, and potential future directions will be discussed.
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http://dx.doi.org/10.1016/j.psj.2019.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587794PMC
February 2020

Predicting Salmonella prevalence associated with meteorological factors in pastured poultry farms in southeastern United States.

Sci Total Environ 2020 Apr 8;713:136359. Epub 2020 Jan 8.

Department of Food Science and Technology, University of Georgia, Athens, GA, USA. Electronic address:

Consumer demand has increased for pastured poultry products as the drive for sustainable farming practices and ethical treatments of livestock have become popular in the press. It is necessary to identify the important meteorological factors associated with the prevalence of Salmonella in the pastured poultry settings since the presence of Salmonella in the environment could lead to contamination of the final product. The objective of this study was to develop a model to describe the relationship between meteorological factors and the presence of Salmonella on the pastured poultry farms. The random forest method was used to develop a model where 83 meteorological factors were included as the predicting variables. The soil model identified humidity as the most important variable associated with Salmonella prevalence, while high wind gust speed and average temperature were identified as important meteorological variables in the feces model. The developed models were robust in predicting the prevalence of Salmonella in pastured poultry farms with the area under receiver operating characteristic (ROC) curve values of 0.884 and 0.872 for the soil model and feces model, respectively. The predictive models developed in this study can provide users with practical and effective tools to make informed decisions with scientific evidence regarding the meteorological parameters that are important to monitor for increased on-farm Salmonella prevalence.
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http://dx.doi.org/10.1016/j.scitotenv.2019.136359DOI Listing
April 2020

Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix).

PeerJ 2019 11;7:e7786. Epub 2019 Oct 11.

Department of Biological Sciences and Museum of Natural Science, Louisiana State University, Baton Rouge, LA, United States of America.

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In , we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.
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http://dx.doi.org/10.7717/peerj.7786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791344PMC
October 2019

A Microbiomic Analysis of a Pasture-Raised Broiler Flock Elucidates Foodborne Pathogen Ecology Along the Farm-To-Fork Continuum.

Front Vet Sci 2019 7;6:260. Epub 2019 Aug 7.

Department of Food Science, Center for Food Safety, University of Arkansas, Fayetteville, AR, United States.

While conventionally grown poultry continues to dominate the U. S. poultry industry, there is an increasing demand for locally-grown, "all natural" alternatives. The use of next generation sequencing allows for not only the gross (e.g., community structure) but also fine-scale (e.g., taxa abundances) examination of these complex microbial communities. This data provides a better understanding of how a pasture flock's microbiome changes throughout the production life cycle and how that change in microbial ecology changes foodborne pathogens in alternative poultry production systems. In order to understand this ecology better, pooled broiler samples were taken during the entire flock life cycle, from pre-hatch gastrointestinal samples ( = 12) to fecal samples from the brood ( = 5), and pasture ( = 10) periods. Additional samples were taken during processing, including skin and feather rinsates ( = 12), ceca ( = 12), and whole carcass rinses ( = 12), and finally whole carcasss rinsates of final products ( = 3). Genomic DNA was extracted, 16S rDNA microbiome sequencing was conducted (Illumina MiSeq), and microbiomes were analyzed and compared using QIIME 1.9.1 to determine how microbiomes shifted throughout production continuum, as well as what environmental factors may be influencing these shifts. Significant microbiome shifts occurred during the life cycle of the pasture broiler flock, with the brood and pasture fecal samples and cecal samples being very distinct from the other pre-hatch, processing, and final product samples. Throughout these varied microbiomes, there was a stable core microbiome containing 13 taxa. Within this core microbiome, five taxa represented known foodborne pathogens () or potential/emerging pathogens () whose relative abundances varied throughout the farm-to-fork continuum, although all were more prevalent in the fecal samples. Additionally, of the 25 physiochemical and nutrient variables measured from the fecal samples, the carbon to nitrogen ratio was one of the most significant variables to warrant further investigations because it impacted both general fecal microbial ecology and and taxa within the core fecal microbiomes. These findings demonstrate the need for further longitudinal, farm-to-fork studies to understand the ecology of the microbial ecology of pasture production flocks to improve animal, environmental, and public health.
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http://dx.doi.org/10.3389/fvets.2019.00260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692657PMC
August 2019

A newly developed Escherichia coli isolate panel from a cross section of U.S. animal production systems reveals geographic and commodity-based differences in antibiotic resistance gene carriage.

J Hazard Mater 2020 01 13;382:120991. Epub 2019 Aug 13.

Bacterial Epidemiology & Antimicrobial Resistance Research Unit, U.S. National Poultry Research Center, Agricultural Research Service, Department of Agriculture, Athens, GA, United States.

There are limited numbers of Escherichia coli isolate panels that represent United States food animal production. The majority of existing Escherichia coli isolate panels are typically designed: (i) to optimize genetic and/or phenotypic diversity; or (ii) focus on human isolates. To address this shortfall in agriculturally-related resources, we have assembled a publicly-available isolate panel (AgEc) from the four major animal production commodities in the United States, including beef, dairy, poultry, and swine, as well as isolates from agriculturally-impacted environments, and other commodity groups. Diversity analyses by phylotyping and Pulsed-field Gel Electrophoresis revealed a highly diverse composition, with the 300 isolates clustered into 71 PFGE sub-types based upon an 80% similarity cutoff. To demonstrate the panel's utility, tetracycline and sulfonamide resistance genes were assayed, which identified 131 isolates harboring genes involved in tetracycline resistance, and 41 isolates containing sulfonamide resistance genes. There was strong overlap in the two pools of isolates, 38 of the 41 isolates harboring sulfonamide resistance genes also contained tetracycline resistance genes. Analysis of antimicrobial resistance gene patterns revealed significant differences along commodity and geographical lines. This panel therefore provides the research community an E. coli isolate panel for study of issues pertinent to U.S. food animal production.
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http://dx.doi.org/10.1016/j.jhazmat.2019.120991DOI Listing
January 2020

Comparison between random forest and gradient boosting machine methods for predicting Listeria spp. prevalence in the environment of pastured poultry farms.

Food Res Int 2019 08 28;122:47-55. Epub 2019 Mar 28.

Department of Food Science and Technology, University of Georgia, Athens, GA, USA. Electronic address:

Foodborne pathogens such as Listeria spp. contain the ability to survive and multiply in poultry farming environments, which provides a route of contamination for poultry processing environments and final poultry products. An understanding of the effect of meteorological variables on the prevalence of Listeria spp. in the farming environment is lacking. Soil and feces samples were collected from 11 pastured poultry farms from 2014 to 2017. Random forest (RF) and gradient boosting machine (GBM) predictive models were generated to describe and predict Listeria spp. prevalence in feces and soil samples based on meteorological factors at the farming location. This study attempted to demonstrate the use of GBM models in a food safety context and compare their use to RF models. Both feces models performed very well, with area under the curve (AUC) values of 0.905 and 0.855 for the RF and GBM models, respectively. The soil GBM model outperformed the RF model with AUCs of 0.873 and 0.700, respectively. The developed models can be used to predict the prevalence of Listeria spp. in pastured poultry farm environments and should be of great use to poultry farmers, producers, and risk managers.
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http://dx.doi.org/10.1016/j.foodres.2019.03.062DOI Listing
August 2019

Applications of Microbiome Analyses in Alternative Poultry Broiler Production Systems.

Front Vet Sci 2019 24;6:157. Epub 2019 May 24.

Center for Food Safety, Food Science Department, University of Arkansas, Fayetteville, AR, United States.

While most of the focus on poultry microbiome research has been directed toward conventional poultry production, there is increasing interest in characterizing microbial populations originating from alternative or non-conventional poultry production. This is in part due to the growing general popularity in locally produced foods and more specifically the attractiveness of free-range or pasture raised poultry. Most of the focus of microbiome characterization in pasture flock birds has been on live bird production, primarily on the gastrointestinal tract. Interest in environmental impacts on production responses and management strategies have been key factors for comparative microbiome studies. This has important ramifications since these birds are not only raised under different conditions, but the grower cycle can be longer and in some cases slower growing breeds used. The impact of different feed additives is also of interest with some microbiome-based studies having examined the effect of feeding these additives to birds grown under pasture flock conditions. In the future, microbiome research approaches offer unique opportunities to develop better live bird management strategies and design optimal feed additive approaches for pasture flock poultry production systems.
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http://dx.doi.org/10.3389/fvets.2019.00157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543280PMC
May 2019

Essential Oils as an Intervention Strategy to Reduce in Poultry Production: A Review.

Front Microbiol 2019 14;10:1058. Epub 2019 May 14.

Center of Food Safety, Department of Food Science, University of Arkansas, Fayetteville, AR, United States.

is a major foodborne pathogen and can be acquired through consumption of poultry products. With 1.3 million United States cases a year, the high prevalence of within the poultry gastrointestinal tract is a public health concern and thus a target for the development of intervention strategies. Increasing demand for antibiotic-free products has led to the promotion of various alternative pathogen control measures both at the farm and processing level. One such measure includes utilizing essential oils in both pre- and post-harvest settings. Essential oils are derived from plant-based extracts, and there are currently over 300 commercially available compounds. They have been proposed to control in the gastrointestinal tract of broilers. When used in concentrations low enough to not influence sensory characteristics, essential oils have also been proposed to decrease bacterial contamination of the poultry product during processing. This review explores the use of essential oils, particularly thymol, carvacrol, and cinnamaldehyde, and their role in reducing concentrations both pre- and post-harvest. This review also details the suggested mechanisms of action of essential oils on .
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http://dx.doi.org/10.3389/fmicb.2019.01058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527745PMC
May 2019

Semi-Quantification of Total and During Egg Incubations Using a Combination of 16S rDNA and Specific Pathogen Primers for qPCR.

Front Microbiol 2018 2;9:2454. Epub 2018 Nov 2.

Department of Food Science, University of Arkansas, Fayetteville, AR, United States.

Rapid molecular techniques that evaluate eggs for the presence of foodborne pathogens is an essential component to poultry food safety monitoring. Interestingly, it is not just table eggs that contribute to outbreaks of foodborne disease. Broiler layer production actively contributes to sustaining of foodborne pathogens within a flock. The surface contamination of production eggs with invasive pathogens such as , , and during embryogenesis results in gastrointestinal tract (GIT) colonization. Pathogens that secure a niche within the GIT during embryonic development are nearly impossible to eradicate from the food chain. Therefore, current monitoring paradigms are not comprehensive because they fail to capture the presence of invasive pathogens within the embryonic GIT rapidly. By developing tools to recognize the pathogens' presence in the GIT during embryogenesis, producers are then able to spot evaluate broiler eggs for their potential risk as carriers of foodborne pathogens. In this study a novel qPCR assay was developed to semi-quantify pathogen load relative to total bacterial burden. Eggs sampled from three independent production broiler flocks of different ages were assayed for , , and against total microbial load (). The eggs were sampled at 1-day post-set within each flock, 2 weeks post-set, after vaccination (at 2.5 weeks) and 1-day post-hatch. The eggs were washed, and the yolk and embryonic chick GIT were collected. The DNA was extracted and subjected to a qPCR assay. The results confirm a novel technique for pathogen monitoring relative to total bacterial load and a unique method for monitoring the dynamics of foodborne pathogen invasion throughout broiler egg production.
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http://dx.doi.org/10.3389/fmicb.2018.02454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6230980PMC
November 2018

Complete Genome Sequence of Listeria monocytogenes Strain MR310, Isolated from a Pastured-Flock Poultry Farm System.

Genome Announc 2018 Mar 8;6(10). Epub 2018 Mar 8.

Department of Food Science and Technology, Oregon State University, Corvallis, Oregon, USA

Investigation of transmission from environmental sources associated with pasture-raised chickens to poultry products is needed to determine ways to prevent potential foodborne illness. Here, we report the complete genome sequence of MR310, one of the isolates from a pastured-flock poultry management system.
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http://dx.doi.org/10.1128/genomeA.00171-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5843743PMC
March 2018

Urea Hydrolysis and Calcium Carbonate Precipitation in Gypsum-Amended Broiler Litter.

J Environ Qual 2018 Jan;47(1):162-169

Broiler () litter is subject to ammonia (NH) volatilization losses. Previous work has shown that the addition of gypsum to broiler litter can increase nitrogen mineralization and decrease NH losses due to a decrease in pH, but the mechanisms responsible for these effects are not well understood. Therefore, three laboratory studies were conducted to evaluate the effect of gypsum addition to broiler litter on (i) urease activity at three water contents, (ii) calcium carbonate precipitation, and (iii) pH. The addition of gypsum to broiler litter increased ammonium concentrations ( < 0.0033) and decreased litter pH by 0.43 to 0.49 pH units after 5 d ( < 0.0001); however, the rate of urea hydrolysis in treated litter only increased on Day 0 for broiler litter with low (0.29 g HO g) and high (0.69 g HO g) water contents, and on Day 3 for litter with medium (0.40 g HO g) water content ( < 0.05). Amending broiler litter with gypsum also caused an immediate decrease in litter pH (0.22 pH units) due to the precipitation of calcium carbonate (CaCO) from gypsum-derived calcium and litter bicarbonate. Furthermore, as urea was hydrolyzed, more urea-derived carbon precipitated as CaCO in gypsum-treated litter than in untreated litter ( < 0.001). These results indicate that amending broiler litter with gypsum favors the precipitation of CaCO, which buffers against increases in litter pH that are known to facilitate NH volatilization.
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http://dx.doi.org/10.2134/jeq2017.08.0337DOI Listing
January 2018

The Distribution of in Pasture-Raised Broiler Farm Soils Is Potentially Related to University of Vermont Medium Enrichment Bias toward over .

Front Vet Sci 2017 21;4:227. Epub 2017 Dec 21.

Egg Safety and Quality Research Unit, U.S. National Poultry Research Center, Agricultural Research Service, United States Department of Agriculture, Athens, GA, United States.

The occurrence of has been widely investigated in the poultry production chain from the processing plant to the final product. However, limited data are available on species, including , in the poultry farm environment. Therefore, fecal and soil samples from 37 pastured poultry flocks from 10 all-natural farms over 3 years were assessed to determine the prevalence and diversity of within these alternative poultry farm environments using standard cultural and molecular methods. species were isolated in 15% of poultry farm samples and included (65.7%), (17.4%), and (15.1%). Additional multiplex PCR serotyping showed group 1/2a-3a to be the most dominant serovar group. Based on these results, monoculture growth experiments were conducted on four soil isolates (three isolates representing the three recovered serovar groups and one isolate) to determine if culture medium [tripticase soy broth (TSB) and University of Vermont modified enrichment broth (UVM)], inoculum concentration (10 or 10 CFU/ml), or incubation temperature (20, 30, and 42°C) differentially affected these species. Overall, very few significant growth differences were observed between the behavior of the three isolates (representing the three recovered serovar groups) under the growth conditions tested. Alternatively, at 30°C in UVM with the lower inoculum concentration, the isolate had a significantly shorter lag phase than the isolates. In coculture growth studies under these same incubation conditions, the lag phase of and was similar, but the final concentration of was significantly higher than . However, cocultures in UVM for high inoculum concentration did not show preferential growth of over . These results indicate that the use of UVM as an enrichment medium may preferentially allow to outcompete at low concentrations, biasing the prevalence from these farm samples toward and potentially underreporting the presence of in these environments.
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http://dx.doi.org/10.3389/fvets.2017.00227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5742580PMC
December 2017

Occurrence in Poultry Flocks: Detection and Potential Implications.

Front Vet Sci 2017 11;4:125. Epub 2017 Aug 11.

Center for Food Safety, Food Science Department, University of Arkansas, Fayetteville, AR, United States.

Foodborne pathogens such as , and are a major concern within the food industry due to their pathogenic potential to cause infection. Of these, , possesses a high mortality rate (approximately 20%) and is considered one of the most dangerous foodborne pathogens. Although the usual reservoirs for transmission have been extensively studied, little is known about the relationship between and live poultry production. Sporadic and isolated cases of listeriosis have been attributed to poultry production and spp. have been isolated from all stages of poultry production and processing. Farm studies suggest that live birds may be an important vector and contributor to contamination of the processing environment and transmission of to consumers. Therefore, the purpose of this review is to highlight the occurrence, incidence, and potential systemic interactions of spp. with poultry.
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http://dx.doi.org/10.3389/fvets.2017.00125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615842PMC
August 2017

Flue-gas desulfurization gypsum effects on urea-degrading bacteria and ammonia volatilization from broiler litter.

Poult Sci 2017 Aug;96(8):2676-2683

USDA-ARS U.S. National Poultry Research Center, 950 College Station Rd., Athens, GA 30605.

A major concern of the broiler industry is the volatilization of ammonia (NH3) from the mixture of bedding material and broiler excretion that covers the floor of broiler houses. Gypsum has been proposed as a litter amendment to reduce NH3 volatilization, but reports of NH3 abatement vary among studies and the mechanism responsible for decreasing NH3 volatilization is not well understood. The goal of this study was to evaluate the effect of adding 20 or 40% flue-gas desulfurization gypsum (FGDG) to broiler litter on pH, electrical conductivity (EC), water potential, urea-degrading bacteria abundance, NH3 and carbon dioxide (CO2) evolution, and nitrogen (N) mineralization in several 21-d experiments. The addition of FGDG to broiler litter increased EC by 24 to 33% (P < 0.0001), decreased urea-degrading bacteria by 48 to 57% (P = 0.0001) and increased N mineralization by 10 to 11% (P = 0.0001) as compared to litters not amended with FGDG. Furthermore, the addition of FGDG to broiler litter decreased NH3 volatilization by 18 to 28% (P < 0.0001), potentially resulting from the significantly lower litter pH values compared to un-amended litter (P < 0.0001). Findings of this study indicate that amending broiler litter with 20% FGDG can decrease NH3 volatilization and increase the fertlizer value of broiler litter.
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http://dx.doi.org/10.3382/ps/pex044DOI Listing
August 2017

In-Package Inactivation of Pathogenic and Spoilage Bacteria Associated with Poultry Using Dielectric Barrier Discharge-Cold Plasma Treatments.

Curr Microbiol 2017 Feb 24;74(2):149-158. Epub 2016 Nov 24.

Poultry Microbiology Safety and Processing Research Unit, US National Poultry Research Center, USDA-ARS, 950 College Station Rd., Athens, GA, 30605, USA.

The goal of this study was to test the efficacy of in-package dielectric barrier discharge-cold plasma (DBD-CP) treatment to inactivate poultry-associated spoilage (Pseudomonas fluorescens) and pathogenic (Salmonella enterica Typhimurium, Campylobacter jejuni) bacteria. Liquid cultures of the bacterial isolates were sealed within packages containing ambient air (Trial 1) or modified air (65% O:30% CO:5% N; Trial 2). The packages were subjected to treatment times ranging from 30 to 180 s, and after 24 h incubation at 4 °C, bacterial titers were determined. The DBD-CP system completely inactivated the four isolates tested, although the in-package gas composition and treatment times were isolate-specific. Both C. jejuni isolates were completely inactivated between 30 s (modified air) and 120 s (ambient air), while modified air was required for the complete inactivation of S. typhimurium (90 s) and P. fluorescens (180 s). This DBD-CP system is effective for inactivating major poultry-associated spoilage and pathogenic bacteria in liquid culture, and through this study, system parameters to optimize inactivation were determined. This study demonstrates the potential for DBD-CP treatment to inactivate major bacteria of economic interest to the poultry industry, thus potentially allowing for reduced spoilage (e.g., longer shelf life) and increased safety of poultry products.
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http://dx.doi.org/10.1007/s00284-016-1158-xDOI Listing
February 2017

Metabolic parameters linked by phenotype microarray to acid resistance profiles of poultry-associated Salmonella enterica.

Res Microbiol 2016 Nov - Dec;167(9-10):745-756. Epub 2016 Jul 12.

University of California, Davis, CA, USA. Electronic address:

Phenotype microarrays were analyzed for 51 datasets derived from Salmonella enterica. The top 4 serotypes associated with poultry products and one associated with turkey, respectively Typhimurium, Enteritidis, Heidelberg, Infantis and Senftenberg, were represented. Datasets were partitioned initially into two clusters based on ranking by values at pH 4.5 (PM10 A03). Negative control wells were used to establish 90 respiratory units as the point differentiating acid resistance from sensitive strains. Thus, 24 isolates that appeared most acid-resistant were compared initially to 27 that appeared most acid-sensitive (24 × 27 format). Paired cluster analysis was also done and it included the 7 most acid-resistant and -sensitive datasets (7 × 7 format). Statistical analyses of ranked data were then calculated in order of standard deviation, probability value by the Student's t-test and a measure of the magnitude of difference called effect size. Data were reported as significant if, by order of filtering, the following parameters were calculated: i) a standard deviation of 24 respiratory units or greater from all datasets for each chemical, ii) a probability value of less than or equal to 0.03 between clusters and iii) an effect size of at least 0.50 or greater between clusters. Results suggest that between 7.89% and 23.16% of 950 chemicals differentiated acid-resistant isolates from sensitive ones, depending on the format applied. Differences were more evident at the extremes of phenotype using the subset of data in the paired 7 × 7 format. Results thus provide a strategy for selecting compounds for additional research, which may impede the emergence of acid-resistant Salmonella enterica in food.
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http://dx.doi.org/10.1016/j.resmic.2016.06.006DOI Listing
March 2017

Antibiotic Resistance Patterns of Major Zoonotic Pathogens from All-Natural, Antibiotic-Free, Pasture-Raised Broiler Flocks in the Southeastern United States.

J Environ Qual 2016 Mar;45(2):593-603

The use of antibiotics in agroecosystems has been implicated in the rise in antibiotic resistance (AR), which can affect environmental, animal, and human health. To determine the environmental impact of antibiotic use in agroecosystems, appropriate background levels of AR in agricultural environments in the absence of antibiotic application must be determined. Therefore, to determine background levels of AR in broiler production, four target microbes (, , , and ) were isolated from 15 all-natural, antibiotic-free, pasture-raised broiler flocks from six farms within the southeastern United States. The AR profiles of these isolates were characterized using the CDC National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS), and these resistance patterns were compared across target microbes and farms and throughout the life cycle of the flocks along the farm-to-fork continuum. Antibiotic resistances were most prevalent in and and least prevalent in . Although and were isolated from the same farms and characterized using the same NARMS plates, they exhibited distinct AR profiles, with demonstrating clear farm-specific resistance patterns. Multidrug resistance rates (three or more antibiotics), in order of prevalence, were (63.9%), (36.0%), (12.7%), and (1.4%). The results of this study demonstrate the variability in background AR among major food safety-related microbes, even when isolated from similar production and processing samples from the same farms, and indicate the need for the proper design of future broiler production studies to account for this highly dynamic background AR.
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http://dx.doi.org/10.2134/jeq2015.07.0366DOI Listing
March 2016

How Should We Be Determining Background and Baseline Antibiotic Resistance Levels in Agroecosystem Research?

J Environ Qual 2016 Mar;45(2):420-31

Although historically, antibiotic resistance has occurred naturally in environmental bacteria, many questions remain regarding the specifics of how humans and animals contribute to the development and spread of antibiotic resistance in agroecosystems. Additional research is necessary to completely understand the potential risks to human, animal, and ecological health in systems altered by antibiotic-resistance-related contamination. At present, analyzing and interpreting the effects of human and animal inputs on antibiotic resistance in agroecosystems is difficult, since standard research terminology and protocols do not exist for studying background and baseline levels of resistance in the environment. To improve the state of science in antibiotic-resistance-related research in agroecosystems, researchers are encouraged to incorporate baseline data within the study system and background data from outside the study system to normalize the study data and determine the potential impact of antibiotic-resistance-related determinants on a specific agroecosystem. Therefore, the aims of this review were to (i) present standard definitions for commonly used terms in environmental antibiotic resistance research and (ii) illustrate the need for research standards (normalization) within and between studies of antibiotic resistance in agroecosystems. To foster synergy among antibiotic resistance researchers, a new surveillance and decision-making tool is proposed to assist researchers in determining the most relevant and important antibiotic-resistance-related targets to focus on in their given agroecosystems. Incorporation of these components within antibiotic-resistance-related studies should allow for a more comprehensive and accurate picture of the current and future states of antibiotic resistance in the environment.
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http://dx.doi.org/10.2134/jeq2015.06.0327DOI Listing
March 2016

Antibiotics in Agroecosystems: Introduction to the Special Section.

J Environ Qual 2016 Mar;45(2):377-93

The presence of antibiotic drug residues, antibiotic resistant bacteria, and antibiotic resistance genes in agroecosystems has become a significant area of research in recent years and is a growing public health concern. While antibiotics are used in both human medicine and agricultural practices, the majority of their use occurs in animal production where historically they have been used for growth promotion, in addition to the prevention and treatment of disease. The widespread use of antibiotics and the application of animal wastes to agricultural lands play major roles in the introduction of antibiotic-related contamination into the environment. Overt toxicity in organisms directly exposed to antibiotics in agroecosystems is typically not a major concern because environmental concentrations are generally lower than therapeutic doses. However, the impacts of introducing antibiotic contaminants into the environment are unknown, and concerns have been raised about the health of humans, animals, and ecosystems. Despite increased research focused on the occurrence and fate of antibiotics and antibiotic resistance over the past decade, standard methods and practices for analyzing environmental samples are limited and future research needs are becoming evident. To highlight and address these issues in detail, this special collection of papers was developed with a framework of five core review papers that address the (i) overall state of science of antibiotics and antibiotic resistance in agroecosystems using a causal model, (ii) chemical analysis of antibiotics found in the environment, (iii) need for background and baseline data for studies of antibiotic resistance in agroecosystems with a decision-making tool to assist in designing research studies, as well as (iv) culture- and (v) molecular-based methods for analyzing antibiotic resistance in the environment. With a focus on the core review papers, this introduction summarizes the current state of science for analyzing antibiotics and antibiotic resistance in agroecosystems, discusses current knowledge gaps, and develops future research priorities. This introduction also contains a glossary of terms used in the core reivew papers of this special section. The purpose of the glossary is to provide a common terminology that clearly characterizes the concepts shared throughout the narratives of each review paper.
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http://dx.doi.org/10.2134/jeq2016.01.0023DOI Listing
March 2016

Subtyping of Salmonella enterica Subspecies I Using Single-Nucleotide Polymorphisms in Adenylate Cyclase.

Foodborne Pathog Dis 2016 07 1;13(7):350-62. Epub 2016 Apr 1.

1 U.S. National Poultry Research Center , U.S. Department of Agriculture, Athens, Georgia .

Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase (cyaA). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer extension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ∼89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilocus sequence typing indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition.
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http://dx.doi.org/10.1089/fpd.2015.2088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939371PMC
July 2016

Recovery of Salmonella enterica serovar Enteritidis from hens initially infected with serovar Kentucky.

Food Chem 2015 Dec 14;189:86-92. Epub 2015 Feb 14.

U.S. Department of Agriculture, 950 College Station Rd., Athens, GA 30605, United States.

Salmonella enterica serovars Enteritidis and Kentucky differ greatly in epidemiology. We wanted to know if the non-pathogenic serotype Kentucky impacted the recovery of the pathogen Enteritidis from chickens. To explore this issue, 4 groups of hens were treated as follows: (i) hens were inoculated orally with Kentucky and injected intramuscularly 2 weeks later with Enteritidis, (ii) hens were contact infected with Kentucky and then with Enteritidis, (iii) hens were injected with Enteritidis only, and (iv) hens were contact infected with Enteritidis only. Hens exposed orally to serotype Kentucky received 10 exp9 CFU, and hens injected with serotype Enteritidis received 10 exp7 CFU intramuscularly. Contact infected hens were kept in rooms with deliberately infected hens. Droppings, cecal tonsils and 5 internal organs were sampled and cultured at 6, 13 and 20 days post-infection from the 4 groups. Egg production was monitored. Results suggest that non-pathogenic serotypes of Salmonella may mitigate recovery of Enteritidis from chickens exposed by contact. In summary, we show results from an initial experiment intended to investigate if multiple serotypes impact the ecology of pathogenic S. enterica on-farm.
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http://dx.doi.org/10.1016/j.foodchem.2015.02.018DOI Listing
December 2015

The characterization of Salmonella enterica serotypes isolated from the scalder tank water of a commercial poultry processing plant: Recovery of a multidrug-resistant Heidelberg strain.

Poult Sci 2015 Mar 12;94(3):467-72. Epub 2015 Feb 12.

U.S. Department of Agriculture, Agricultural Research Service, Poultry Microbiological Safety and Processing Research Unit, 950 College Station Road, Athens, GA 30605.

The recent multistate outbreak of a multidrug-resistant (MDR) Salmonella Heidelberg strain from commercial poultry production highlights the need to better understand the reservoirs of these zoonotic pathogens within the commercial poultry production and processing environment. As part of a larger study looking at temporal changes in microbial communities within the major water tanks within a commercial processing facility, this paper identifies and characterizes Salmonella enterica isolated from the water in a final scalder tank at 3 times during a typical processing day: prior to the birds entering the tank (start), halfway through the processing day (mid), and after the final birds were scalded (end). Over 3 consecutive processing days, no Salmonella were recovered from start-of-day water samples, while a total of 56 Salmonella isolates were recovered from the mid-day and end-of-day scalder water samples. Traditional and newer PCR-based serotyping methods eventually identified these isolates as either group C3 S. Kentucky (n=45) and group B S. Heidelberg (n=11). While none of the S. Kentucky isolates possessed any resistances to the antimicrobials tested, all S. Heidelberg isolates were found to be multidrug resistant to 5 specific antimicrobials representing 3 antimicrobial classes. Due to the potential public health impact of S. Heidelberg and the recent nationwide poultry-associated outbreak of multidrug-resistant S. Heidelberg, future studies should focus on understanding the transmission and environmental growth dynamics of this serotype within the commercial poultry processing plant environment.
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http://dx.doi.org/10.3382/ps/peu060DOI Listing
March 2015

A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples.

J Vis Exp 2014 Dec 10(94). Epub 2014 Dec 10.

Department of Biological Sciences, Center for Microbial Genetics and Genomics, Northern Arizona University.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the "gold standard" enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.
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http://dx.doi.org/10.3791/52161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396950PMC
December 2014