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Abstract Hatcheries, where eggs from multiple breeder farms are incubated and hatched before being sent to different broiler farms, represent a nexus point in the commercial production of broilers in the United States. Considering all downstream microbial quality and safety aspects of broiler production (live production, processing, consumer use) can be potentially affected by the hatchery, a better understanding of microbial ecology within commercial hatcheries is essential. Therefore, a commercial broiler hatchery was biomapped using 16S rRNA amplicon-based microbiome analyses of four sample type categories (Air, Egg, Water, Facility) across five different places in the pre-hatch, hatch, and post-hatch areas. While distinct microbiota were found for each sample type category and hatchery area, microbial community analyses revealed that Egg microbiota trended towards clustering with the facility-related samples when moving from the prehatch to post-hatch areas, highlighting the potential effect of the hatchery environment in shaping the pre-harvest broiler-related microbiota. Prevalence analyses revealed 20 ASVs (Core20) present in the core microbiota of all sample types and areas, with each ASV possessing a unique distribution throughout the hatchery. Interestingly, three Enterobacteriaceae ASVs were in the Core20, including Salmonella. Subsequent analyses showed that Salmonella, while a minor prehatch and hatch Core20ASV, dominated the Enterobacteriaceae niche and total microbiota in the chick pad feces in the post-hatch area of the hatchery, and the presence of this Salmonella ASV in the post-hatch feces was associated with swabs of breakroom tables. These findings highlight the complexity of commercial hatchery microbiota, including identifying chick pad feces and breakroom tables as potentially important sampling or disinfection targets for hatchery managers to focus their Salmonella mitigation efforts to reduce loads entering live production farms.

Poultry-associated salmonellosis results in significant costs to poultry producers and consumers. Given the vertically integrated nature of the United States poultry industry, a better understanding of Salmonella ecology throughout all levels of poultry production is essential. One nexus point is the hatchery, where eggs from multiple broiler breeder farms are incubated and hatched, with the chicks being sent to numerous farms; therefore, the hatchery represents an ideal area to understand preharvest Salmonella ecology and flow. To achieve this, a commercial broiler hatchery was biomapped, focusing on Salmonella prevalence and serotype diversity among four major sample type categories (Air, Egg, Water, Facility) across five different places in the prehatch, hatch, and posthatch areas. Following two sets of eggs from broiler breeder farms over two production days, the overall Salmonella prevalence was 26% (48/184). Of the positive samples, the highest prevalence was observed in swabs taken from the floor drains in the facility and transport truck (56%), as well as in the hatch and posthatch hatchery areas (50%). Kentucky (n = 17), Gaminara (n = 12), and Alachua (n = 11) were the dominant Salmonella serotypes, with serotypes of greatest outbreak concern from chickens (Enteritidis) representing only 6.25% (3/48) of all recovered Salmonella isolates. The posthatch transport area, including the underfloor reservoirs of the transport trucks, not only harbored Enteritidis but also the enrichment broths from these Salmonella-positive samples also possessed sequences matching the commercial live-attenuated vaccine Typhimurium strain according to CRISPR SeroSeq analyses. These findings highlight the complex diversity of commercial hatchery Salmonella populations, including identifying facility floor drains and transport trucks as potentially important critical control points for hatchery managers to focus their Salmonella mitigation efforts to reduce loads and serotypes entering live production farms.

There is an increasing need to develop alternative antimicrobials to replace currently used antibiotics. Phytochemicals, such as essential oils, have garnered significant attention in recent years as potential antimicrobials. However, the mechanisms underlying their bactericidal activities are not yet fully understood. In this study, we investigated the bactericidal activity of eugenol oil against Salmonella enterica serovar Typhimurium (S. Typhimurium) to elucidate its mechanism of action. We hypothesized that eugenol exerts its bactericidal effects through the production of reactive oxygen species (ROS), which ultimately leads to cell death. The result of this study demonstrated that the bactericidal activity of eugenol against S. Typhimurium was significantly (p < 0.05) mitigated by thiourea (ROS scavenger) or iron chelator 2,2′-dipyridyl, supporting the hypothesis. This finding contributes to a better understanding of the killing mechanism by eugenol oil.

Abstract Background Microbiomes that can serve as an indicator of gut, intestinal, and general health of humans and animals are largely influenced by food consumed and contaminant bioagents. Microbiome studies usually focus on estimating the alpha (within sample) and beta (similarity/dissimilarity among samples) diversities. This study took a combinatorial approach and applied machine learning to microbiome data to predict the presence of disease-causing pathogens and their association with known/potential probiotic taxa. Probiotics are beneficial living microorganisms capable of improving the host organism’s digestive system, immune function and ultimately overall health. Here, 16 S rRNA gene high-throughput Illumina sequencing of temporal pre-harvest (feces, soil) samples of 42 pastured poultry flocks (poultry in this entire work solely refers to chickens) from southeastern U.S. farms was used to generate the relative abundance of operational taxonomic units (OTUs) as machine learning input. Unique genera from the OTUs were used as predictors of the prevalence of foodborne pathogens (Salmonella, Campylobacter and Listeria) at different stages of poultry growth (START (2–4 weeks old), MID (5–7 weeks old), END (8–11 weeks old)), association with farm management practices and physicochemical properties. Result While we did not see any significant associations between known probiotics and Salmonella or Listeria, we observed significant negative correlations between known probiotics (Bacillus and Clostridium) and Campylobacter at the mid-time point of sample collection. Our data indicates a negative correlation between potential probiotics and Campylobacter at both early and end-time points of sample collection. Furthermore, our model prediction shows that changes in farm operations such as how often the houses are moved on the pasture, age at which chickens are introduced to the pasture, diet composition and presence of other animals on the farm could favorably increase the abundance and activity of probiotics that could reduce Campylobacter prevalence. Conclusion Integration of microbiome data with farm management practices using machine learning provided insights on how to reduce Campylobacter prevalence and transmission along the farm-to-fork continuum. Altering management practices to support proliferation of beneficial probiotics to reduce pathogen prevalence identified here could constitute a complementary method to the existing but ineffective interventions such as vaccination and bacteriophage cocktails usage. Study findings also corroborate the presence of bacterial genera such as Caloramator, DA101, Parabacteroides and Faecalibacterium as potential probiotics.

In this study, we explore how transformer models, which are known for their attention mechanisms, can improve pathogen prediction in pastured poultry farming. By combining farm management practices with microbiome data, our model outperforms traditional prediction methods in terms of the F1 score—an evaluation metric for model performance—thus fulfilling an essential need in predictive microbiology. Additionally, the emphasis is on making our model’s predictions explainable. We introduce a novel approach for identifying feature importance using the model’s attention matrix and the PageRank algorithm, offering insights that enhance our comprehension of established techniques such as DeepLIFT. Our results showcase the efficacy of transformer models in pathogen prediction for food safety and mark a noteworthy contribution to the progress of explainable AI within the biomedical sciences. This study sheds light on the impact of effective farm management practices and highlights the importance of technological advancements in ensuring food safety.

Abstract Impaired gut homeostasis is associated with stroke often presenting with leaky gut syndrome and increased gut, brain, and systemic inflammation that further exacerbates brain damage. We previously reported that intracisternal administration of Tanshinone IIA-loaded nanoparticles (Tan IIA-NPs) and transplantation of induced pluripotent stem cell-derived neural stem cells (iNSCs) led to enhanced neuroprotective and regenerative activity and improved recovery in a pig stroke model. We hypothesized that Tan IIA-NP + iNSC combination therapy-mediated stroke recovery may also have an impact on gut inflammation and integrity in the stroke pigs. Ischemic stroke was induced, and male Yucatan pigs received PBS + PBS (Control, n = 6) or Tan IIA-NP + iNSC (Treatment, n = 6) treatment. The Tan IIA-NP + iNSC treatment reduced expression of jejunal TNF-α, TNF-α receptor1, and phosphorylated IkBα while increasing the expression of jejunal occludin, claudin1, and ZO-1 at 12 weeks post-treatment (PT). Treated pigs had higher fecal short-chain fatty acid (SCFAs) levels than their counterparts throughout the study period, and fecal SCFAs levels were negatively correlated with jejunal inflammation. Interestingly, fecal SCFAs levels were also negatively correlated with brain lesion volume and midline shift at 12 weeks PT. Collectively, the anti-inflammatory and neuroregenerative treatment resulted in increased SCFAs levels, tight junction protein expression, and decreased inflammation in the gut.

Despite the continuous progress in food science and technology, the global burden of foodborne illnesses remains substantial, with pathogens in food causing millions of infections each year. Traditional microbiological culture methods are inadequate in detecting the full spectrum of these microorganisms, highlighting the need for more comprehensive detection strategies. This review paper aims to elucidate the relationship between foodborne pathogen colonization and the composition of the poultry microbiome, and how this knowledge can be used for improved food safety. Our review highlights that the relationship between pathogen colonization varies across different sections of the poultry microbiome. Further, our review suggests that the microbiome profile of poultry litter, farm soil, and farm dust may serve as potential indicators of the farm environment's food safety issues. We also agree that the microbiome of processed chicken samples may reveal potential pathogen contamination and food quality issues. In addition, utilizing predictive modeling techniques on the collected microbiome data, we suggest establishing correlations between particular taxonomic groups and the colonization of pathogens, thus providing insights into food safety, and offering a comprehensive overview of the microbial community. In conclusion, this review underscores the potential of microbiome analysis as a powerful tool in food safety, pathogen detection, and risk assessment.

AimCampylobacter is the leading bacterial pathogen that causes foodborne illnesses worldwide. Pasture farming is regarded as an important source of agricultural production for small farming communities. Consumer preference for pasture-raised animal products has increased; however, there is a paucity of information on the microbiological quality of pasture-raised poultry products. The purpose of this study was to explore genetic relatedness of thermophilic Campylobacter isolates, to assess antibiotic resistance phenotypically and genotypically, and to screen the presence of virulence determinants of Campylobacter isolates from pasture-raised poultry farms from southeastern United States.MethodsNinety-seven Campylobacter isolates previously identified by Q7 BAX® System Real-Time PCR were genotyped by multilocus sequence typing (MLST). Campylobacter isolates were then evaluated for their phenotypic antimicrobial susceptibility against nine antimicrobial agents using Sensititre plates. Additionally, Campylobacter isolates were tested for the presence of antimicrobial resistance-associated elements. Furthermore, Campylobacter isolates were screened for the presence of 13 genes encoding putative virulence factors by PCR. These included genes involved in motility (flaA and flhA), adhesion and colonization (cadF, docC, racR, and virB11), toxin production (cdtA, cdtB, cdtC, wlaN, and ceuE) and invasion (ciaB and iamA).ResultsAmong 97 Campylobacter isolates, Campylobacter jejuni (n = 79) and Campylobacter coli (n = 18) were identified. By MLST, C. jejuni isolates were assigned to seven clonal complexes. Among them, ST-353, ST-607 and ST-21 were the most common STs recognized. All C. coli (n = 18) isolates were included in CC-828. Interestingly, eight STs identified were not belonging any previous identified clonal complex. Campylobacter isolates displayed a high resistance rate against tetracycline (81.4%), while a low rate of resistance was observed against macrolides (azithromycin and erythromycin), quinolones and fluoroquinolones (nalidixic acid and ciprofloxacin), aminoglycosides (gentamicin), ketolide (telithromycin), amphenicol (florfenicol) and lincomycin (clindamycin). Thirteen isolates (13.54%) were pan-susceptible to all tested antibiotics, while nine isolates were multi-antimicrobial resistant (MAR; resist to three or more antimicrobial classes). Interestingly, there were no isolates resistant to all antimicrobial classes. Thr86Ile mutation was identified in all quinolones resistant strains. Erythromycin encoding gene (ermB) was identified in 75% of erythromycin resistant isolates. The A2075 mutation was detected in one erythromycin resistant strain, while A2074 could not be identified. The tetO gene was identified in 93.7% of tetracycline resistant isolates and six tetracycline susceptible isolates. In conclusion, the results of this study revealed that Campylobacter isolates from pasture-raised poultry farms showed the ST relatedness to Campylobacter isolates commonly associated with humans, indicating pasture-raised broiler flocks, similar to conventionally-reared broiler flocks, as a potential vector for antibiotic-resistant and pathogenic strains of thermophilic Campylobacter to humans.

The catabolic activity of the ruminal microbial community of cattle enables the conversion of low-quality feedstuffs into meat and milk. The rate at which this conversion occurs is termed feed efficiency, which is of crucial importance given that feed expenses account for up to 70% of the cost of animal production. The present study assessed the relationship between cattle feed efficiency and the composition of their ruminal microbial communities during the feedlot finishing period. Angus steers (n = 65) were fed a feedlot finishing diet for 82 days and their growth performance metrics were evaluated. These included the dry matter intake (DMI), average daily gain (ADG), and residual feed intake (RFI). Steers were rank-ordered based upon their RFI, and the five lowest RFI (most efficient) and five highest RFI (least efficient) steers were selected for evaluations. Ruminal fluid samples were collected on days 0 and 82 of the finishing period. Volatile fatty acids (VFA) were quantified, and microbial DNA was extracted and the 16S rRNA gene was sequenced. The results showed that the ADG was not different (p = 0.82) between efficiency groups during the 82-day feedlot period; however, the efficient steers had lower (p = 0.03) DMI and RFI (p = 0.003). Less-efficient (high RFI) steers developed higher (p = 0.01) ruminal Methanobrevibacter relative abundances (p = 0.01) and tended (p = 0.09) to have more Methanosphaera. In high-efficiency steers (low RFI), the relative abundances of Ruminococcaceae increased (p = 0.04) over the 82-day period. The molar proportions of VFA were not different between the two efficiency groups, but some changes in the concentration of specific VFA were observed over time. The results indicated that the ruminal microbial populations of the less-efficient steers contained a greater relative abundance of methanogens compared to the high-efficiency steers during the feedlot phase, likely resulting in more energetic waste in the form or methane and less dietary energy being harvested by the less-efficient animals.

Food additives are employed in the food industry to enhance the color, smell, and taste of foods, increase nutritional value, boost processing efficiency, and extend shelf life. Consumers are beginning to prioritize food ingredients that they perceive as supporting a healthy lifestyle, emphasizing ingredients they deem acceptable as alternative or “clean-label” ingredients. Ready-to-eat (RTE) meat products can be contaminated with pathogens and spoilage microorganisms after the cooking step, contributing to food spoilage losses and increasing the risk to consumers for foodborne illnesses. More recently, consumers have advocated for no artificial additives or preservatives, which has led to a search for antimicrobials that meet these demands but do not lessen the safety or quality of RTE meats. Lactates and diacetates are used almost universally to extend the shelf life of RTE meats by reducing spoilage organisms and preventing the outgrowth of the foodborne pathogen Listeria monocytogenes. These antimicrobials applied to RTE meats tend to be broad-spectrum in their activities, thus affecting overall microbial ecology. It is to the food processing industry’s advantage to target spoilage organisms and pathogens specifically.

Abstract Background Campylobacter jejuni is the leading bacterial pathogen that causes foodborne illness worldwide. Because of genetic diversity and sophisticated growth requirements of C. jejuni, several genotyping methods have been investigated to classify this bacterium during the outbreaks. One of such method is to use clustered regularly interspaced short palindromic repeats (CRISPR). Objectives The goal of this study was to explore the diversity of C. jejuni isolates with CRISPR from an animal farm. Methods Seventy‐seven C. jejuni isolates from an animal farm were used in this study. The day‐old broilers were reared with other poultry and farm animals, including layer hens, guinea hens, dairy goats and sheep. A small swine herd was also present on an adjacent, but separate plot of land. Isolation and identification of C. jejuni were performed according to the standard procedures. The CRISPR type 1 was PCR amplified from genomic DNA, and the amplicons were sequenced by the Sanger dideoxy method. The direct repeats (DRs) and spacers of the CRISPR sequences were identified using the CRISPRFinder. Results The CRISPR sequences were detected in all 77 isolates. One type of DRs was identified in these 77 isolates. The lengths of the CRISPR locus ranged from 100 to 560 nucleotides, whereas the number of spacers ranged from one to eight. The distributions of the numbers of CRISPR spacers from different sources seemed to be random. Overall, 17 out of 77 (22%) C. jejuni isolates had two and five spacers, whereas 14 out of 77 (18%) isolates had three spaces in their genomes. By further analysis of spacer sequences, a total of 266 spacer sequences were identified in 77 C. jejuni isolates. By comparison with known published spacer sequences, we observed that 49 sequences were unique in this study. The CRISPR sequence combination of Nos. 16, 19, 48 and 57 was found among a total of 15 C. jejuni isolates containing various multi‐locus sequence typing (MLST) types (ST‐50, ST‐607, ST‐2231 and ST‐5602). No. 57 spacer sequence was unique from this study, whereas the other three (Nos. 16, 19 and 48) sequences were found in previous reports. Combination of Nos. 5, 9, 15, 30 and 45 was associated with ST‐353. To compare the CRISPR genotyping with other methods, the MLST was selected due to its high discriminatory power to differentiate isolates. Based on calculation of the Simpson's index of diversity, a combination of both methods had higher Simpson's index value than those for CRISPR or MLST, respectively. Conclusions Our results suggest that the MLST from C. jejuni isolates can be discriminated based on the CRISPR unique spacer sequences and the numbers of spacers. In the future, investigation on the CRISPR resolution for C. jejuni identification in outbreaks is needed. A database that integrates both MLST sequences and CRISPR sequences and is searchable is greatly in demand for tracking outbreaks and evolution of this bacterium.

Animal sourced foods including contaminated poultry meat and eggs contribute to human non-typhoidal salmonellosis, a foodborne zoonosis. Prevalence of Salmonella in pastured poultry production systems can lead to contamination of the final product. Identification of farm practices that affect Salmonella prevalence is critical for implementing control measures to ensure the safety of these products. In this study, we developed predictive models based predominantly on deep learning approaches to identify key pre-harvest management variables (using soil and feces samples) in pastured poultry farms that contribute to Salmonella prevalence. Our ensemble approach utilizing five different machine learning techniques predicts that physicochemical parameters of the soil and feces (elements such as sodium (Na), zinc (Zn), potassium (K), copper (Cu)), electrical conductivity (EC), the number of years that the farms have been in use, and flock size significantly influence pre-harvest Salmonella prevalence. Egg source, feed type, breed, and manganese (Mn) levels in the soil/feces are other important variables identified to contribute to Salmonella prevalence on larger (≥3 flocks reared per year) farms, while pasture feed and soil carbon-to-nitrogen ratio are predicted to be important for smaller/hobby (

Listeria continues to be a persistent foodborne pathogen that is responsible for human cases of listeriosis when contaminated food products are consumed. Human subjects considered to be most susceptible include the elderly, immunocompromised, and pregnant women. Listeria is characterized as a saprophytic organism with the capability of responding and adapting to constantly changing environments because it possesses multiple stress response mechanisms to overcome varying temperatures, salt concentrations, and pH, among others. Primary foods and food products associated with listeriosis include dairy products and ready-to-eat meats such as turkey products. Historically, chicken eggs have not been identified as a primary source of Listeria, but the potential for contamination during egg production and processing does exist. Listeria species have been isolated from egg-processing plant equipment and are presumed to occur in egg-processing plant environments. Whether Listeria is consistently disseminated onto eggs beyond the egg-processing plant is a risk factor that remains to be determined. However, research has been conducted over the years to develop egg wash solutions that generate combinations of pH and other properties that would be considered inhibitory to Listeria. Even less is known regarding the association of Listeria with alternative egg production systems, but Listeria has been isolated from pasture flock broilers, so it is conceivable, given the nature of the outdoor environments, that layer birds under these conditions would also be exposed to Listeria and their eggs become contaminated. This review focuses on the possibility of Listeria occurring in conventional and alternative egg-laying production and processing systems.

Campylobacter has consistently posed a food safety issue in broiler meat. This study aimed to create a quantitative microbial risk assessment model from retail to consumption, designed to evaluate the seasonal risk of campylobacteriosis associated with broiler meat consumption in the United States. To achieve this, data was gathered to build distributions that would enable us to predict the growth of Campylobacter during various stages such as retail storage, transit, and home storage. The model also included potential fluctuations in concentration during food preparation and potential cross-contamination scenarios. A Monte Carlo simulation with 100,000 iterations was used to estimate the risk of infection per serving and the number of infections in the United States by season. In the summer, chicken meat was estimated to have a median risk of infection per serving of 9.22 × 10−7 and cause an average of about 27,058,680 infections. During the winter months, the median risk of infection per serving was estimated to be 4.06 × 10−7 and cause an average of about 12,085,638 infections. The risk assessment model provides information about the risk of broiler meat to public health by season. These results will help understand the most important steps to reduce the food safety risks from contaminated chicken products.

ABSTRACT: The use of “omics” has become widespread across poultry production, from breeding to management to bird health to food safety and everywhere in between. While the conventional poultry industry has become more exposed to the power and utility of “omic” technologies, smaller poultry flock producers typically do not have this same level of experience. Because smaller, nonconventional poultry production is a growing portion of the overall poultry market, it is important that they also have educational access to these research tools and the resultant data. While small flock producers are dedicated and knowledgeable farmers, their knowledge of these newer technologies may be limited at best, and it is the task of academic researchers to communicate the importance of these “omic” tools and how the omic data can improve a variety of different aspects of their operations. This review discusses ways to effectively communicate complex microbiota and microbial genome sequence data to small flock producers and transforming this data into meaningful and applicable information that they can utilize to inform beneficial management decisions.

Campylobacter is a major foodborne pathogen with over a million United States cases a year and is typically acquired through the consumption of poultry products. The common occurrence of Campylobacter as a member of the poultry gastrointestinal tract microbial community remains a challenge for optimizing intervention strategies. Simultaneously, increasing demand for antibiotic-free products has led to the development of several alternative control measures both at the farm and in processing operations. Bacteriophages administered to reduce foodborne pathogens are one of the alternatives that have received renewed interest. Campylobacter phages have been isolated from both conventionally and organically raised poultry. Isolated and cultivated Campylobacter bacteriophages have been used as an intervention in live birds to target colonized Campylobacter in the gastrointestinal tract. Application of Campylobacter phages to poultry carcasses has also been explored as a strategy to reduce Campylobacter levels during poultry processing. This review will focus on the biology and ecology of Campylobacter bacteriophages in poultry production followed by discussion on current and potential applications as an intervention strategy to reduce Campylobacter occurrence in poultry production.

(1) Background: Foodborne illness from Salmonella enterica subspecies I is most associated with approximately 32 out of 1600 serotypes. While whole genome sequencing and other nucleic acid-based methods are preferred for serotyping, they require expertise in bioinformatics and often submission to an external agency. Intergenic Sequence Ribotyping (ISR) assigns serotype to Salmonella in coordination with information freely available at the National Center for Biotechnology Information. ISR requires updating because it was developed from 26 genomes while there are now currently 1804 genomes and 1685 plasmids. (2) Methods: Serotypes available for sequencing were analyzed by ISR to confirm primer efficacy and to identify any issues in application. Differences between the 2012 and 2022 ISR database were tabulated, nomenclature edited, and instances of multiple serotypes aligning to a single ISR were examined. (3) Results: The 2022 ISR database has 268 sequences and 40 of these were assigned new NCBI accession numbers that were not previously available. Extending boundaries of sequences resolved hdfR cross-alignment and reduced multiplicity of alignment for 37 ISRs. Comparison of gene cyaA sequences and some cell surface epitopes provided evidence that homologous recombination was potentially impacting results for this subset. There were 99 sequences that still had no match with an NCBI submission. (4) The 2022 ISR database is available for use as a serotype screening method for Salmonella enterica subspecies I. Finding that 36.9% of the sequences in the ISR database still have no match within the NCBI Salmonella enterica database suggests that there is more genomic heterogeneity yet to characterize.

Greater consumer demand for all natural, antibiotic-free poultry products has led to an increase in pastured poultry operations. Given the increased level of environmental interaction, and the potential increase in exposure to foodborne pathogens, a greater understanding of the prevalence and diversity of Salmonella populations inherent within pastured poultry flocks. To achieve this, 42 pastured poultry flocks from 11 farms were sampled using a farm-to-fork strategy and Salmonella was isolated and characterized through pre-harvest (feces, soil) to post-harvest (ceca, whole carcass rinse) to the final product (whole carcass rinse) the consumer would purchase. Salmonella was isolated from 353 of a total of 2,305 samples, representing an overall prevalence of 18.1%. By far the most prevalent serotype was Kentucky (72.7% of all isolates), with

ABSTRACT: 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.

The reported increase in antibiotic-resistant bacteria in humans has resulted in a major shift away from antibiotic use in food animal production. This shift has been driven by the assumption that removing antibiotics will select for antibiotic susceptible bacterial taxa, which in turn will allow the currently available antibiotic arsenal to be more effective.

ABSTRACT: 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.

ABSTRACT: 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.

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 (P = 0.04) in the group of piglets fed carbadox. Faith's phylogenetic diversity was similar (P = 0.89) for both groups of piglets on day 35, but it was diminished (P = 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 (P = 0.27) in both groups on day 63. Likewise, abundances of Slackia, Peptococcus, Catenibacterium, Coprococcus, and Blautia were all similar between the two groups (P ≥ 0.40) on day 35, but were smaller in the carbadox group (P ≤ 0.05) on day 49; however, on day 63, abundances of all these genera were once again similar (P ≥ 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 (P ≥ 0.52), but decreased in the carbadox group on day 49 (P ≤ 0.05), and were similar again in both groups on day 63 (P ≥ 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.

Due to nutritional benefits and perceived humane ways of treating the animals, the demand for antibiotic-free pastured poultry chicken has continued to be steadily rise. Despite the non-usage of antibiotics in pastured poultry broiler production, antibiotic resistance (AR) is reported in zoonotic poultry pathogens. However, factors that drive multidrug resistance (MDR) in pastured poultry are not well understood. In this study, we used machine learning and deep learning approaches to predict farm management practices and physicochemical properties of feces and soil that drive MDR in zoonotic poultry pathogens. Antibiotic use in agroecosystems is known to contribute to resistance. Evaluation of the development of resistance in environments that are free of antibiotics such as the all-natural, antibiotic-free, pastured poultry production systems described here is critical to understand the background AR in the absence of any selection pressure, i.e., basal levels of resistance. We analyzed 1635 preharvest (feces and soil) samples collected from forty-two pastured poultry flocks and eleven farms in the Southeastern United States. CDC National Antimicrobial Resistance Monitoring System guidelines were used to determine antimicrobial/multidrug resistance profiles of Salmonella, Listeria, and Campylobacter. A combination of two traditional machine learning (RandomForest and XGBoost) and three deep learning (Multi-layer Perceptron, Generative Adversarial Network, and Auto-Encoder) approaches identified critical farm management practices and environmental variables that drive multidrug resistance in poultry pathogens in broiler production systems that represents background resistance. This study enumerates management practices that contribute to AR and makes recommendations to potentially mitigate multidrug resistance and the prevalence of Salmonella and Listeria in pastured poultry.

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 Lactobacillus 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.

Antimicrobial resistance is an ancient bacterial defense mechanism that has rapidly spread due to the frequent use of antibiotics for disease treatment and livestock growth promotion. We are becoming increasingly aware that pathogens, such as members of the genus Acinetobacter, are precipitously evolving drug resistances through multiple mechanisms, including the acquisition of antibiotic resistance genes. In this study, we isolated three multidrug resistant Acinetobacter species from birds on a free-range farm. Acinetobacter radioresistens, Acinetobacter lwoffii, and Acinetobacter johnsonii were isolated from hens, turkeys and ducks and were resistant to 14 clinically relevant antibiotics, including several listed by the World Health Organization as essential medicines. Co-culturing any of the three Acinetobacter species with Acinetobacter baumannii resulted in contact-dependent release of intact resistance determinants. We also isolated several lytic bacteriophages and selected two of these phages to be included in this study based on differences in plaquing characteristics, nucleic acid content and viral morphology. Both phages released host DNA, including antibiotic resistance genes during cell lysis and we demonstrated that these resistance determinants were transferable to a naïve strain of Escherichia coli. This study demonstrates that contact-dependent competition between bacterial species can readily contribute to DNA release into the environment, including antibiotic resistance determinants. We also highlight that the constant lysis and turnover of bacterial populations during the natural lifecycle of a lytic bacteriophage is an underappreciated mechanism for the liberation of DNA and subsequent genetic exchange.

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, Dickeya dadantii DSM 18020, as a commercially utilized pectinase producer in poultry diets against other known pectinase producers, in silico. In the current study, whole genomes of Dickeya dadantii DSM 18020 (Dd18020), D. dadantii 3937 (Dd3937), D. solani IPO 2222 (Ds2222), Bacillus halodurans C-125 (BhC125), and B. subtilis subsp. subtilis 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 Bacillus 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 in vitro and determining the stability of the produced pectinases during feed manufacturing are necessary.

The gastrointestinal microbiota of cattle is important for feedstuff degradation and feed efficiency determination. This study evaluated the fecal microbiome of Angus steers with distinct feed efficiencies during the feedlot-finishing phase. Angus steers (n = 65), fed a feedlot-finishing diet for 82 days, had growth performance metrics evaluated. Steers were ranked based upon residual feed intake (RFI), and the 5 lowest RFI (most efficient) and 5 highest RFI (least efficient) steers were selected for evaluation. Fecal samples were collected on 0-d and 82-d of the finishing period and microbial DNA was extracted and evaluated by 16S rRNA gene sequencing. During the feedlot trial, inefficient steers had decreased (p = 0.02) Ruminococcaceae populations and increased (p = 0.01) Clostridiaceae populations. Conversely, efficient steers had increased Peptostreptococcaceae (p = 0.03) and Turicibacteraceae (p = 0.01), and a trend for decreased Proteobacteria abundance (p = 0.096). Efficient steers had increased microbial richness and diversity during the feedlot period, which likely resulted in increased fiber-degrading enzymes in their hindgut, allowing them to extract more energy from the feed. Results suggest that cattle with better feed efficiency have greater diversity of hindgut microorganisms, resulting in more enzymes available for digestion, and improving energy harvest in the gut of efficient cattle.

Diet impacts the composition of the ruminal microbiota; however, prior to slaughter, cattle are fasted, which may change the ruminal microbial ecosystem structure and lead to dysbiosis. The objective of this study was to determine changes occurring in the rumen after pre-slaughter fasting, which can allow harmful pathogens an opportunity to establish in the rumen. Ruminal samples were collected before and after pre-slaughter fasting from seventeen commercial Angus steers. DNA extraction and 16S rRNA gene sequencing were performed to determine the ruminal microbiota, as well as volatile fatty acid (VFA) concentrations. Microbial richness (Chao 1 index), evenness, and Shannon diversity index all increased after fasting (p ≤ 0.040). During fasting, the two predominant families Prevotellaceae and Ruminococcaceae decreased (p ≤ 0.029), whereas the remaining minor families increased (p < 0.001). Fasting increased Blautia and Methanosphaera (p ≤ 0.003), while Campylobacter and Treponema tended to increase (p ≤ 0.086). Butyrate concentration tended to decrease (p = 0.068) after fasting. The present findings support that fasting causes ruminal nutrient depletion resulting in dysbiosis, allowing opportunistic pathogens to exploit the void in the ruminal ecological niche.

Adenine and thymine homopolymer strings of at least 8 nucleotides (AT 8+mers) were characterized in Salmonella enterica subspecies I. The motif differed between other taxonomic classes but not between Salmonella enterica serovars. The motif in plasmids was possibly associated with serovar. Approximately 12.3% of the S. enterica motif loci had mutations. Mutability of AT 8+mers suggests that genomes undergo frequent repair to maintain optimal gene content, and that the motif facilitates self-recognition; in addition, serovar diversity is associated with plasmid content. A theory that genome regeneration accounts for both persistence of predominant Salmonella serovars and serovar diversity provides a new framework for investigating root causes of foodborne illness.

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 Adapterama I, 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 Adapterama 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.

Nutrient and pathogen pollution are the leading causes of water quality impairment in lakes, reservoirs, and rivers in the United States. Dissemination of these contaminants can result in eutrophication of freshwater resources and pose a risk to public health through recreational contact and degradation of waters used as drinking water sources. Agricultural production practices, both crop and animal, have been identified as sources of excess nutrients and microbial pathogens contributing to freshwater pollution. In the U.S., commercial meat poultry production has been targeted as a source of both excess nutrients, especially phosphorus, and fecal indicator bacteria (FIB) in regional watersheds. Recently, there has been an increase in pastured poultry operations where chickens have access to fresh pasture on a daily basis. However, few studies have explored the environmental sustainability of these types of poultry production systems. In the case of pastured poultry systems in close proximity to watersheds, there is a need to better understand potential environmental impacts in order to implement sustainable and cost-effective practices. The identification of such environmental and economic benefits would complement the mission and objectives of farmers using pastured poultry production systems and may add more value to their product. This review will focus on potential mitigation strategies to enhance environmental sustainability and provide economic benefit to small scale pastured poultry operations.

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 (N = 12) to fecal samples from the brood (N = 5), and pasture (N = 10) periods. Additional samples were taken during processing, including skin and feather rinsates (N = 12), ceca (N = 12), and whole carcass rinses (N = 12), and finally whole carcasss rinsates of final products (N = 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 (Salmonella, Campylobacter) or potential/emerging pathogens (Pseudomonas, Enterococcus, Acinetobacter) 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 Campylobacter and Enterococcus 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.

Rice is one of the most economically important foods in the world today. The FAO has reported that managing rice processing and the resulting byproducts into more sustainable applications would be beneficial for a variety of reasons. Rice processing involves several milling stages to produce edible final products. The milling process is the most important step in rice production because it determines the nutritional, cooking, and sensory qualities of crude rice. As crude rice goes through the milling process, byproducts are generated, such as bran that have been shown to exhibit beneficial impacts on human and animal nutrition. While several rice byproducts have applications in agriculture, rice bran has probably received the most attention for its functional properties. Rice bran is a mixture of protein, fat, ash, and crude fiber. However, rice bran's composition is largely dependent on the type of rice and the efficiency of the milling system. Based on studies with mice, rice bran has been shown to elicit prebiotic-like properties by preventing colonization of Salmonella in the gastrointestinal tract. More recently, in vitro incubation studies with chicken cecal contents have demonstrated that certain rice varieties are more inhibitory to Salmonella than others. Moreover, the byproducts of the rice milling process can also provide an economic boost for rice producing nations. In this review, the byproducts of the milling process, how they are utilized, and potential application for rice milling byproducts are discussed.

The poultry farm environment plays a key role in the microbial colonization of chickens during production, which shapes what enters the processing and final retail environments. The aim of this study was to evaluate the effect of the farm environment on the microbial composition of pasture-raised broilers using a combined cultural and microbiomic farm-to-fork. To achieve this, two nearby pastured poultry farms raised small flocks of Freedom Ranger broilers obtained from the same hatchery flock and fed the same diet throughout live production. The major differences between the two farms were the physical farm environment, the method of feather removal during processing (scalding vs. skinning), and the storage conditions of carcasses before customers received final product (refrigeration vs. freezing). Microbiomes were compared from fecal and soil samples (live production), ceca (processing), and whole carcass rinses (processing, final product) to determine what effect the physical farm environment had on the poultry-related microbiomes. Overall, microbiomes in feces (p < 0.04), soil (p < 0.02), and ceca (p < 0.02) samples from farm 1 harbored a higher taxonomic richness than farm 2. Beta-diversity analysis demonstrated significant differences between the broiler microbiomes of the two farms for samples collected at the live production (p < 0.04) and processing stages (p < 0.01), but not for final product carcass rinses. At the early live production stage (~3 weeks old), fecal microbiomes from farm 1 were positively correlated to aluminum, iron, manganese, silicon and zinc concentrations in feces but not fecal microbiota from farm 2. At the late live production stage (~12 weeks old), fecal microbiomes from both farms were no longer correlated to mineral content of feces but were negatively correlated to fecal pH. Given that the farm environment itself was the major difference, the results show that even when raising the same breed fed the same diet, poultry farms have their own ecology that shape the composition of the poultry-related microbiomes. Therefore, it is vital that future work focuses on elucidating the farm environmental variables that have the greatest influence on these microbiomes, thus allowing for eventual targeted interventions to better manage these microbial populations to benefit animal, environmental, and public health.

Campylobacter 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 Campylobacter 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 Campylobacter 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 Campylobacter concentrations both pre- and post-harvest. This review also details the suggested mechanisms of action of essential oils on Campylobacter.

Listeria monocytogenes is a psychrotrophic Gram positive organism that is considered one of the more critical foodborne pathogens of public health concern. To prevent illness the USDA and FDA enforce a zero-tolerance policy for Listeria on ready-to-eat foods such as delicatessen meats and poultry. Regardless, L. monocytogenes can still be isolated from food production facilities and retail products, indicating that current sanitation methods are not always sufficient. Both conventional and alternative poultry production and processing systems have also been identified as potential sources of Listeria spp. Concerns associated with alternative poultry production and processing can be further exacerbated by limitations on sanitation and available antimicrobials for usage in organic and natural poultry products. Furthermore, mobile poultry processing units often process organic and small-scale poultry farms that are not able to be processed by conventional standing facilities. These alternative production facilities and their products are often exempt from federal inspection, due to processing a relatively low number of carcasses. Due to these exemptions, it is unknown if sufficient sanitation is applied in these alternative processing facilities to prevent L. monocytogenes contamination. Organic processing restrictions may also impact which sanitizers and antimicrobials can be utilized. This review describes variations between conventional and mobile poultry processing units in conjunction with how L. monocytogenes may persist in the processing environment and on retail products. This review will also examine alternative antimicrobials proven to be effective against Listeria spp. and potentially be acceptable for use in alternative poultry production systems.

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.

The succession of bacterial species as birds mature can impact their growth efficiency, health, and food safety due to the ability of a mature gastrointestinal microbial population to exclude opportunistic pathogenic bacteria from colonization. There has been an increase in the number of chickens fed a soy-free diet compared to the standard soybean-containing rations in pastured poultry. Therefore, the present study was designed to describe the impacts of feeding a soy-containing (SOY) or a soy-free diet (SF) on the bacterial succession in the gut of pasture-raised chickens. In order to compare microbiome compositions as birds aged, 16s rRNA gene sequencing was performed on the gastrointestinal tract of 1-day-old birds, on fecal material obtained at different ages, and on the cecal contents collected at slaughter from broilers fed each of the 2 diets. Results showed that at the phylum level, regardless of diet and birds' stage of life, there was a predominance of the phylum Firmicutes, which ranged from 50 to 90% of all OTUs in all samples. Moreover, the number of observed OTUs significantly increased (P < 0.001) as birds in the SF diet grew older; however, no significant variations (P = 0.11) in this trait were observed as birds in the SOY diet aged. Shannon Diversity indices calculated for birds in both diets had greater values (P < 0.001) in the cecal contents of adult birds collected at slaughter, compared to samples obtained at earlier ages. Also in spite of which diet was offered, the abundance of Oscillospira, Faecalibacterium, Ruminococcus, and 2 unidentified genera from the orders Clostridiales and RF39 all increased as birds aged, while abundance of Lactobacillus decreased. The predicted metabolic functions using microbiome data showed no differences (P ≥ 0.26) in metabolism of amino acids, carbohydrates, lipids, and energy for both diets. These findings demonstrate that, under these experimental conditions, while diet modulates some microbial changes in the gut of pasture-raised chicken, overall, a similar successional pattern is observed for both diets. Moreover, the resulting metabolic functions are very similar between diets regardless of age.

Soybean is one of the primary ingredients in poultry diets, but it causes problems in some consumers with allergies. Thus, production of poultry without soybean in their diets has increased in recent years. In addition, consumers are increasingly supporting alternative (e.g., organic, pasture-raised) chicken production systems for ethical reasons, yet the impacts of a variety of diets on these birds remain unclear. The present study was designed to examine the impact of feeding two different diets—a soybean-based and a soy-free diet—on the intestinal microbiome of pasture-raised chickens. 16S rRNA gene sequencing was performed on the feces, cecal contents, and whole carcass rinses from 5 pasture-raised flocks (3 soybean-based, 2 soy-free) grown over 2 years on the same farm, and their microbiomes were compared. Regardless of diet, the phylum Firmicutes corresponded to 63% or more of the total bacterial abundance in the fecal and cecal samples; however, in the whole carcass rinses, the relative abundance of Firmicutes dropped to ~30%. Alpha diversity metrics revealed significant differences without any clear patterns when comparing the 2 diets, but principal coordinate analysis of β-diversity showed significant differences (P ≤ 0.04) between them. Notably, broilers receiving the soy-free diet had lower abundance of Campylobacter during their entire lifecycle. This effect was particularly important in the fecal material collected when birds were 12-weeks-old (i.e., day of processing; P = 0.003) and in the whole carcass rinses obtained from the final product (P = 0.04). Abundance of Acinetobacter was also lower (P = 0.05) in the whole carcass rinses from birds consuming the soy-free diet. These data suggest that the presence of soybeans in chicken diets can be an important factor shaping overall microbiomes throughout the farm-to-fork continuum, specifically resulting in a greater presence of the foodborne pathogens Campylobacter and Acinetobacter. Therefore, the use of a soy-free diet can potentially represent a viable strategy to reduce contamination of carcasses in pasture-raised chicken production systems.

Predictive models offer food scientists, farmers, and processors tools to help identify variables that lead to an increase in the food safety risk of a product. Foodborne pathogens, such as Listeria spp., pose a major problem for the pastured poultry industry. Currently, there is a lack of understanding of what farm practices lead to higher prevalence of Listeria spp. This study constructed random forest (RF) models to predict the prevalence of Listeria spp. in pastured poultry farming environments and the final broiler product based on major farm practices and variables. Feces, soil, and whole carcass rinse samples were collected from 11 farms in the southeastern United States and evaluated for Listeria spp. presence. The preharvest sample RF model identified the time of year and age of the broiler flock at time of sampling as factors of increased probability of Listeria spp. presence in feces and soil samples. The final product RF model identified brood feed and the presence of chlorine in processing rinse water as the two most important variables associated with an increased likelihood of Listeria spp. presence. Both the preharvest RF model and final sample RF model performed well on a held-out test set, with area under the receiver operating characteristic curve values of 0.876 and 0.887, respectively. The presented models showed the usefulness of RF models in a food safety context. Both RF models will help pastured poultry farmers and processors guide control strategies to manage Listeria contamination in pastured poultry farms and products.

The objective of this study was to investigate the effects of in-package dielectric barrier discharge (DBD) atmospheric cold plasma (CP) on meat color, microbiological quality and safety of chicken breast meat (pectoralis major). Raw broiler breast meat was collected from a local commercial plant. Noninoculated meat samples and meat samples inoculated with Campylobacter and Salmonella were packed in polymeric trays with air. The packaged samples were CP-treated at 70 kV for different times (0, 60, 180, or 300 sec) and stored at 4°C for 5 days. Microbial counts (psychrophiles, Campylobacter, Salmonella) and meat color (International Commission on Illumination (CIE) L∗a∗b∗) were measured before CP treatments and after 5 days of posttreatment storage. Psychrophile growth was inhibited (P0.05) in a∗ and b∗ values between pretreatment and posttreatment plus storage; however, all CP treatments resulted in increased L∗ value (P

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 Salmonella enterica, Campylobacter jejuni, and Listeria monocytogenes 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 S. enterica (invA), C. jejuni (HipO), and L. monocytogenes (HlyA) against total microbial load (16s). 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.

The occurrence of Listeria monocytogenes has been widely investigated in the poultry production chain from the processing plant to the final product. However, limited data are available on Listeria species, including Listeria monocytogenes, 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 Listeria within these alternative poultry farm environments using standard cultural and molecular methods. Listeria species were isolated in 15% of poultry farm samples and included Listeria innocua (65.7%), L. monocytogenes (17.4%), and Listeria welshimeri (15.1%). Additional multiplex PCR serotyping showed group 1/2a-3a to be the most dominant L. monocytogenes serovar group. Based on these results, monoculture growth experiments were conducted on four Listeria soil isolates (three L. monocytogenes isolates representing the three recovered serovar groups and one L. innocua isolate) to determine if culture medium [tripticase soy broth (TSB) and University of Vermont modified Listeria enrichment broth (UVM)], inoculum concentration (102 or 105 CFU/ml), or incubation temperature (20, 30, and 42°C) differentially affected these Listeria species. Overall, very few significant growth differences were observed between the behavior of the three L. monocytogenes isolates (representing the three recovered serovar groups) under the growth conditions tested. Alternatively, at 30°C in UVM with the lower inoculum concentration, the L. innocua isolate had a significantly shorter lag phase than the L. monocytogenes isolates. In coculture growth studies under these same incubation conditions, the lag phase of L. innocua and L. monocytogenes was similar, but the final concentration of L. innocua was significantly higher than L. monocytogenes. However, cocultures in UVM for high inoculum concentration did not show preferential growth of L. innocua over L. monocytogenes. These results indicate that the use of UVM as an enrichment medium may preferentially allow L. innocua to outcompete L. monocytogenes at low concentrations, biasing the Listeria prevalence from these farm samples toward L. innocua and potentially underreporting the presence of L. monocytogenes in these environments.

Foodborne pathogens such as Salmonella, Campylobacter, Escherichia coli, and Listeria are a major concern within the food industry due to their pathogenic potential to cause infection. Of these, Listeria monocytogenes, possesses a high mortality rate (approximately 20%) and is considered one of the most dangerous foodborne pathogens. Although the usual reservoirs for Listeria transmission have been extensively studied, little is known about the relationship between Listeria and live poultry production. Sporadic and isolated cases of listeriosis have been attributed to poultry production and Listeria 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 Listeria to consumers. Therefore, the purpose of this review is to highlight the occurrence, incidence, and potential systemic interactions of Listeria spp. with poultry.