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The standardization of the microbiome sequencing of poultry rinsates is essential for generating comparable microbial composition data among poultry processing facilities if this technology is to be adopted by the industry. Samples must first be acquired, DNA must be extracted, and libraries must be constructed. In order to proceed to library sequencing, the samples should meet quality control standards. Finally, data must be analyzed using computer bioinformatics pipelines. This data can subsequently be incorporated into more advanced computer algorithms for risk assessment. Ultimately, *a uniform sequencing pipeline will enable both the government regulatory agencies and the poultry industry to identify potential weaknesses in food safety.This chapter presents the different steps for monitoring the population dynamics of the microbiome in poultry processing using 16S rDNA sequencing.

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Traditionally, surveillance programs for food products and food processing environments have focused on targeted pathogens and resistance genes. Recent advances in high throughput sequencing allow for more comprehensive and untargeted monitoring. This study assessed the microbiome and resistome in a poultry burger processing line using culturing techniques and whole metagenomic sequencing (WMS). Samples included meat, burgers, and expired burgers, and different work surfaces. Microbiome analysis revealed spoilage microorganisms as the main microbiota, with substantial shifts observed during the shelf-life period. Core microbiota of meat and burgers included Pseudomonas spp., Psychrobacter spp., Shewanella spp. and Brochothrix spp., while expired burgers were dominated by Latilactobacillus spp. and Leuconostoc spp. Cleaning and disinfection (C&D) procedures altered the microbial composition of work surfaces, which still harbored Hafnia spp. and Acinetobacter spp. after C&D. Resistome analysis showed a low overall abundance of resistance genes, suggesting that effective interventions during processing may mitigate their transmission. However, biocide resistance genes were frequently found, indicating potential biofilm formation or inefficient C&D protocols. This study demonstrates the utility of combining culturing techniques and WMS for comprehensive of the microbiome and resistome characterization in food processing lines.

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The formation of Campylobacter jeuni biofilms on processing surfaces is a significant concern in poultry processing, contributing to food safety risks. This study focused on assessing the biofilm forming capabilities of 12 field isolates of C. jejuni of different aerotolerance categories on stainless steel surfaces, a prevalent material in poultry processing environments. Working cultures of each isolate were prepared to approximately 6 log CFU/mL and incubated on stainless steel coupons under microaerobic or aerobic conditions at room temperature or 42°C for 72 h. Biofilm attached cells were enumerated using direct plating and biofilm density was measured using a crystal violet assay by measuring the optical density (OD600) a. Data analysis was conducted using the PROC GLIMMIX procedure in SAS 9.4 with a significance level of 0.05. The study revealed a notable interaction between aerotolerance categories and temperature (P < 0.039) impacting the number of biofilms attached C. jejuni cells on stainless steel coupons. All isolates had significantly higher counts when incubated at 42°C compared to room temperature, regardless of oxygen level (P < 0.001). Furthermore, stronger biofilm density was observed at 42°C compared to room temperature, regardless of oxygen level. These findings underscore the influence of temperature on the biofilm forming ability of C. jejuni. The ability of these field isolates to form biofilms under various environmental conditions suggests a heightened potential for surface colonization and increased infection risk in poultry processing facilities.

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Chicken and chicken products have been associated with foodborne pathogens such as Salmonella, Campylobacter, and Escherichia coli (E. coli). Poultry comprises an important segment of the agricultural economy (75 million birds processed as of 2019) in West Virginia (WV). The risk of pathogens on processed chickens has risen with the increased popularity of mobile poultry processing units (MPPUs). This study evaluated the microbial safety of broilers processed in a MPPU in WV. This study assessed aerobic plate counts (APCs), E. coli counts and the presence/absence of Salmonella and Campylobacter on 96 broiler carcasses following each MPPU step of scalding, eviscerating, and chilling. Samples were either chilled in ice water only (W) or ice water with 5 ppm chlorine (CW). The highest number of bacteria recovered from carcasses were APCs (4.21 log10CFU/mL) and E. coli (3.77 log10CFU/mL; P = 0.02). A total reduction of 0.30 (P = 0.10) and 0.63 (P = 0.01) log10CFU/mL for APCs and E. coli, respectively, occurred from chilling carcasses in CW. Overall, results show that E. coli, Salmonella, and Campylobacter were significantly (P < 0.05) reduced from the initial scalding to the chilling step. However, Salmonella frequency doubled (15.63-34.38%) after the evisceration step, indicating that washing carcasses after evisceration may be a critical control point in preventing cross-contamination by Salmonella. Proper chilling is also an important microbial mitigation step in MPPU processing. Results indicate that Campylobacter was more resistant to chilling than Salmonella. Campylobacter was not completely inactivated until carcasses were chilled in CW, whereas W was sufficient to reduce Salmonella on carcasses. The results led to the conclusion that although 5 ppm chlorine (Cl2) achieved more bacterial reductions than water alone, the reductions were not always significant (P > 0.05). Further MPPU studies are needed to verify more effective chilling and processing strategies.

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We explored the potential of machine learning to identify significant genes associated with Salmonella stress response during poultry processing using whole genome sequencing (WGS) data. The Salmonella isolates (n = 177) used in this study were obtained from various chicken sources (skin before chiller, chicken carcass before chiller, frozen chicken, and post-chill chicken carcass). Six machine learning algorithms (random forest, neural network, cost-sensitive learning, logit boost, and support vector machine linear and radial kernels) were trained on Salmonella WGS data, and model fit was assessed using standard evaluation metrics such as the area under the receiver operating characteristic (AUROC) curve and confusion matrix statistics. All models achieved high performances based on the AUROC metric, with logit boost showing the best performance with an AUROC score of 0.904, sensitivity of 0.889, and specificity of 0.920. The significant genes identified included ybtX, which encodes a Yersiniabactin-associated zinc transporter, and the transferase-encoding genes yccK and thiS. Additionally, genes coding for cold (cspA, cspD, and cspE) and heat shock (rpoH and rpoE) responses were identified. Other significant genes included those involved in lipopolysaccharide biosynthesis (irp1, waaD, rfc, and rfbX), DNA repair and replication (traI), biofilm formation (ccdA and fyuA), and cellular metabolism (irtA).

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In pursuit of sustainability, we explored replacing conventional dissolved air floatation (DAF) in poultry processing wastewater (PPW) treatment with a precisely tuned 0.02 µm stainless-steel ultrafiltration (SSUF) membrane. SSUF is a robust, homogenously porous membrane with strong chemical resistance, ease of cleaning, and exceptional resistance to organic fouling. Unlike polymeric membranes, it can be regenerated multiple times, making it a cost-effective choice due to its compatibility with harsh chemical cleaning. The PPW used for the study was untreated wastewater from all processing units and post-initial screening. Our study revealed the SSUF membrane's exceptional efficiency at eliminating contaminants. It achieved an impressive removal rate of up to 99.9% for total suspended solids (TSS), oil, grease, E. coli, and coliform. Additionally, it displayed a notable reduction in chemical oxygen demand (COD), biochemical oxygen demand (BOD), and total Kjeldahl nitrogen (TKN), up to 90%, 76%, and 76%, respectively. Our investigation further emphasized the SSUF membrane's ability in pathogen removal, affirming its capacity to effectively eradicate up to 99.99% of E. coli and coliform. The measured critical flux of the membrane was 48 Lm-2h-1 at 38 kPa pressure and 1.90 m/s cross-flow velocity. In summary, our study highlights the considerable potential of the SSUF membrane. Its robust performance treating PPW offers a promising avenue for reducing its environmental impact and advocating for sustainable wastewater management practices.

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Measuring circularity is necessary to prove the feasibility of transforming linear technologies into circular ones. However, most of the circular economic researches consider water only as a medium. Food industry processes are excellent examples of systems that are hard to break free from linearity, albeit not impossible. This paper explores solutions to include water in circularity calculations using a Hungarian poultry processing plant as a case study. Two circular economic indicators, the questionnaire-type Circular Economy Indicator Prototype (CEIP) and the product-centric Material Circularity Indicator (MCI and MCI') and the Water Footprint were examined in detail and modified to fit the needs of assessing circularity with water included as raw material. The calculations were supported by Life Cycle Assessment (LCA). The impact on circularity and the environment were quantified by considering different reuse scenarios. As the results of CEIP show, including water reuse in the technology or recycling for irrigation could increase the indicator values from low to medium-high level of circularity. However, the level of improvement highly depends on the amount of water used. LCA highlighted the significant environmental effects of packaging (<2% of product mass) and the relative benefits of recycling and reuse. The MCI' values (including water as raw material) increased from 0.171 to 0.848 when water demand was reduced by 50% and 100% reused within the processes. This led to a reduction of 76% in the environmental effect. On the other hand, Water Footprint analysis showed that 99% of the water is incorporated in the product itself; therefore, technological water consumption should be treated separately from broiler breeding. The results show that a fairly linear process can be directed towards circularity. However, environmental benefits are not guaranteed with higher circularity points, and recycling may lead to unexpected results.

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This study characterized biofilm formation of various Salmonella strains on common processing plant surface materials (stainless steel, concrete, rubber, polyethylene) under static and fluidic shear stress conditions. Surface-coupons were immersed in well-plates containing 1 mL of Salmonella (6 log CFU/mL) and incubated aerobically for 48 h at 37 °C in static or shear stress conditions. Biofilm density was determined using crystal violet assay, and biofilm cells were enumerated by plating on tryptic soy agar plates. Biofilms were visualized using scanning electron microscopy. Data were analyzed by SAS 9.4 at a significance level of 0.05. A surface-incubation condition interaction was observed for biofilm density (p < 0.001). On stainless steel, the OD600 was higher under shear stress than static incubation; whereas, on polyethylene, the OD600 was higher under static condition. Enumeration revealed surface-incubation condition (p = 0.024) and surface-strain (p < 0.001) interactions. Among all surface-incubation condition combinations, the biofilm cells were highest on polyethylene under fluidic shear stress (6.4 log/coupon; p < 0.001). Biofilms of S. Kentucky on polyethylene had the highest number of cells (7.80 log/coupon) compared to all other strain-surface combinations (p < 0.001). Electron microscopy revealed morphological and extracellular matrix differences between surfaces. Results indicate that Salmonella biofilm formation is influenced by serotype, surface, and fluidic shear stress.

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Bio-mapping studies play an important role, as the data collected can be managed and analyzed in multiple ways to look at process trends, find explanations about the effect of process changes, activate a root cause analysis for events, and even compile performance data to demonstrate to inspection authorities or auditors the effect of certain decisions made on a daily basis and their effects over time in commercial settings not only from the food safety perspective but also from the production side. This study presents an alternative analysis of bio-mapping data collected throughout several months in a commercial poultry processing operation as described in the article "Bio-Mapping Indicators and Pathogen Loads in a Commercial Broiler Processing Facility Operating with High and Low Antimicrobial Interventions". The conducted analysis identifies the processing shift effect on microbial loads, attempts to find correlation between microbial indicators data and pathogens loads, and identifies novel visualization approaches and conducts distribution analysis for microbial indicators and pathogens in a commercial poultry processing facility. From the data analyzed, a greater number of locations were statistically different between shifts under reduced levels of chemical interventions with higher means at the second shift for both indicators and pathogens levels. Minimal to negligible correlation was found when comparing aerobic counts and Enterobacteriaceae counts with Salmonella levels, with significant variability between sampling locations. Distribution analysis and visualization as a bio-map of the process resulted in a clear bimodality in reduced chemical conditions for multiple locations mostly explained by shift effect. The development and use of bio-mapping data, including proper data visualization, improves the tools needed for ongoing decision making in food safety systems.

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Peracetic acid (PAA) applied to whole poultry carcasses can reduce the number of Campylobacter, a leading cause of human gastroenteritis. However, previous modelling experiments indicated that Campylobacter survived in greater numbers when pre-treated with a thermal stress equivalent to poultry processing scalding prior to chilling with PAA than when subject to chilling with PAA only. To better understand how Campylobacter responds to PAA, proteomes of C. jejuni poultry strain 2704 were measured after exposure to PAA (60 ppm, pH 4.0) for 45 min under laboratory ambient conditions (approximately 23 °C) to establish a foundational map of survival mechanism before combining with other stresses. Analysis of 580 quantified proteins did not indicate a triggered "peroxide shock" response, nor were common heat shock responses detected. Thioredoxin, iron homeostatic, peroxiredoxins and cytochrome c peroxidases became more abundant suggesting that PAA disturbed cytoplasmic redox homeostasis resulting in antioxidant activation and increased prioritisation of iron homeostasis. The PAA treatment led to responses that included an increased priority for oxidative phosphorylation and a simultaneous decrease in central metabolism associated protein abundances. Lon protease was induced suggesting it has a role in maintaining homeostasis during non-thermal stress. Proteins in flagella and chemotaxis became more abundant though whether PAA has a chemorepellent effect requires further investigation. Overall, the proteome data suggests there was a rapid cellular response to applied PAA stress in the first 15 min with the adaptation to the stress completing between 30 and 45 min. The findings will help guide PAA implementation in commercial poultry processing in terms of processing location and length of application.

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The intensification of the poultry industry may lead to the increased spread of antibiotic resistance genes (ARGs) in the environment. However, the impacts of wastewater discharge from poultry processing plants on the sediment resistome are relatively unexplored. Furthermore, its relationships with important biogeochemical pathways, such as the N cycle, are virtually unknown. The overall objective of this study was to examine the abundance and diversity of antibiotic resistance and N cycling genes in sediment microbial communities impacted by poultry industry wastewater. We performed a metagenomic investigation of sediments in an impacted and a reference tidal creek. We also quantified the abundance of the clinical class 1 integron-integrase gene (intI1) through qPCR as a secondary marker of anthropogenic contamination. Abundance and diversity of ARGs were substantially higher in the impacted tidal creek, especially near the wastewater discharge. Abundances of ARGs conferring resistance to macrolides, tetracyclines, and streptogramins were also higher in the impacted creek than the reference creek. From the N cycling genes detected in the metagenomes, nrfA, the genetic marker for dissimilatory nitrate reduction to ammonia (DNRA), had the strongest positive relationship with the total abundance of ARGs, which may indicate an increased potential of eutrophication in ARG-impacted ecosystems due to nitrogen retention. This study demonstrates that wastewater discharge from a poultry processing plant can increase the spread of ARGs, which may result in negative impacts on ecosystem health.

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Campylobacteriosis is the most commonly notified foodborne disease in New Zealand and poultry meat is the major source for human infection. Carcasses and portions were sampled from key points along primary and secondary processing chains of three New Zealand poultry processors to determine the impact of processing steps on Campylobacter concentrations. Primary processing reduced Campylobacter concentrations on carcasses by almost 6-log; the biggest reduction was achieved by the spinchill, followed by the scald step. Significant plant differences in the degree of Campylobacter reduction were also observed at these steps. The spinchill and final acidified sodium chlorite wash resulted in carcasses with low-to-no levels of Campylobacter regardless of concentrations at prior steps. A similar study was conducted at primary processing for one plant in 2013; significant improvements in Campylobacter mitigation since 2013 were noted. Campylobacter concentrations from final product from secondary processing were higher than concentrations at the end of primary processing. Drumsticks had lower Campylobacter concentrations than other portion types. Skin removal from product did not consistently result in product with lower Campylobacter concentrations. Results identify key areas to target for further reduction of Campylobacter on poultry meat, and provide a benchmark to compare the efficacy of future interventions.

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The importance of thermotolerant Campylobacter spp. as a food-borne pathogen continues to increase and there is a great need for rapid quantitative results in routine diagnostics. However, currently, only the culture-based ISO method is authorized for use in the context of official food control. The present study therefore aimed to assess the suitability of a qPCR method for a rapid quantitative determination of Campylobacter spp. at different stages in the poultry production chain and its equivalence with the culture-based method. Samples from two processors were collected and evaluated both separately and together. Censored regression (Tobit) models have been used to establish a relationship between Campylobacter qPCR counts on the carcasses and explanatory variables of processor and meat counts. Further, correlations of qPCR Campylobacter spp. counts at the different stages of production were calculated. In addition, the comparative data between microbiological enumeration and qPCR results were statistically analyzed. In the correlation calculation of the qPCR results, a highly significant relationship between the Campylobacter spp. counts of the neck skin samples to breast fillet and leg samples could be calculated, indicating a good prediction of Campylobacter spp. loads in these samples. The intercalating dye ethidium monoazide (EMA) was used to see whether the correlations between microbiological counts and qPCR results were improved by pretreating fecal and cecal samples before qPCR analysis. It was shown that the observed values of scatter plots between the qPCR-based and the culture-based methods were strongly correlated. However, on average, the qPCR results were two log10 CFU/mL levels higher than the microbiological counts. However, the classical culture-based method for food hygiene risk assessment cannot be replaced one-to-one by the qPCR or EMA-qPCR. The qPCR method can rather be used for the rapid identification of particularly highly contaminated flocks.

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In poultry processing, Salmonella and Campylobacter contaminations are major food safety concerns. Peracetic acid (PAA) is an antimicrobial commonly used in commercial poultry processing to reduce pathogen prevalence so as to meet the USDA-FSIS performance standards. The objective of this study was to determine the prevalence of Salmonella and Campylobacter on broiler meat in various steps of commercial poultry processing in plants that use PAA. Post-pick, pre-chill, post-chill, and drumstick chicken samples were collected from three processing plants and mechanically deboned meat (MDM) was collected from two of the three plants. Each plant was sampled thrice, and 10 samples were collected from each processing step during each visit. Among the 420 samples, 79 were contaminated with Salmonella and 155 were contaminated with Campylobacter. Salmonella and Campylobacter contamination on the post-pick samples averaged 32.2%. Significant reductions in Salmonella and Campylobacter were observed in pre-chill to post-chill samples, where the prevalence was reduced from 34% and 64.4% to nondetectable limits and 1.1%, respectively (p < 0.001). Salmonella and Campylobacter remained undetectable on the drumstick samples in all three processing plants. However, the prevalence of Salmonella and Campylobacter on MDM was similar to the post-pick prevalence, which suggests substantial cross-contamination from post-chill to MDM.

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The occurrence of egg proteins in products containing spent fowl manufactured under current practices was studied to assess the risk these food products may pose to egg-allergic consumers and to determine if Precautionary Allergen Labelling (PAL) was recommended. Spent fowl slaughtering and processing operations in 2 Canadian facilities were observed. Raw hen pieces (n = 134), coming from 2 facilities, and intermediate and processed products containing spent fowl (n = 57), coming from one facility, were analyzed using ELISA. All samples tested positive for egg proteins. Raw pieces were tested using a qualitative method (i.e., swabbing); estimated egg proteins concentrations suggest the presence of highly contaminated samples (>600 mg/kg in 2 hen wing samples). Swabbing was found to be efficient for rapid detection of eggs in raw hen pieces, but not for quantification. A comparison between swab and grind results showed that egg proteins concentration is underestimated by at least a factor 2 for whole carcasses and a factor 10 for breast, wings and drumsticks, when using the swab protocol. For intermediate and processed products, quantitative measurements indicate that egg protein levels were below 16 mg/kg. Additionally, 88 water samples from chiller tanks were analyzed and indicate that this step could be the cause of the global contamination observed with an increase in egg protein concentrations overtime during the production schedule. As egg contamination is not adequately controlled under the current good production practices, the use of PAL would be recommended for raw spent fowl products.

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In poultry processing, spoilage microbes are persistent microorganisms, which affect the quality of broiler meat. Peracetic acid (PAA) is the most common antimicrobial used by commercial processing plants, which can reduce the prevalence of these microbes. The goal of this study was to determine the concentrations of aerobic bacteria, coliforms, lactic acid bacteria, and Pseudomonas on broiler meat in processing plants that use peracetic acid in various concentrations as the primary antimicrobial. Samples were collected from three processing plants at five processing steps: post-pick (defeathering), pre-chill, post-chill, mechanically deboned meat (MDM), and drumsticks. Samples were rinsed in buffered peptone water for bacteria isolation. Over six log CFU/sample of aerobic plate counts (APC), lactic acid bacteria, and coliforms were detected on post-pick samples. All spoilage bacteria were reduced to nondetectable levels on post-chill samples (p < 0.001). However, the presence of all bacteria on mechanically deboned meat (MDM) samples indicated varying degrees of cross contamination from post-chill and MDM samples. These results suggest PAA effectively reduces spoilage microbes in chilling applications irrespective of differences in PAA concentrations. However, due to the levels of spoilage microbes detected in MDM, it may be worth investigating the potential interventions for this stage of processing.

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Poultry processing is undergoing changes both in operations as well as microbial methodologies. Traditionally, microbial data has been gathered through a series of culturing methods using liquid media and plating for isolation and enumeration. Both foodborne pathogens and nonpathogenic bacterial populations are estimated to assess food safety risks as well as the potential for spoilage. Bacterial loads from carcasses are important for estimating processing control and the effectiveness of antimicrobial applications. However, these culture-based approaches may only provide part of the microbial ecology landscape associated with chicken carcasses and the subsequent changes that occur in these populations during processing. Newer molecular-based approaches, such as 16S sequencing of the microbiota, offer a means to retrieve a more comprehensive microbial compositional profile. However, such approaches also result in large data sets which must be analyzed and interpreted. As more data is generated, this will require not only bioinformatic programs to process the data but appropriate educational forums to present the processed data to a broad audience.

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This study aimed to analyze the incidence of Campylobacter in a small-scale chicken meat processing plant producing "chicken-sashimi", and determine the effectiveness of surface burning as a treatment during processing. The most probable number (MPN) method was used to analyze the load of Campylobacter in 48 samples from four different processing steps (de-feathering, chilling, surface burning, and final-products; 12 samples each). We found the highest load of isolated bacteria in chicken skin after de-feathering. Campylobacter was not detected after the surface burning step despite a large load of bacteria present in the cecum content. Campylobacter was absent in the final products. Adequate surface burning can avoid Campylobacter contamination of chicken sashimi in the processing plant by applying the external stripping method.

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Human infection with Chlamydia psittaci is rare but difficult to recognize. We report an outbreak of psittacosis among poultry processing workers in China. We applied metagenomic next-generation sequencing and identified Chlamydia psittaci reads from samples of all seven patients, four of which were subsequently confirmed by PCR. Epidemiological results suggested that the poultry processed in the factory was the possible source of human infection.