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ARS Home » Southeast Area » Fayetteville, Arkansas » Poultry Production and Product Safety Research » Research » Research Project #439695

Research Project: Multi-hurdle Approaches for Controlling Foodborne Pathogens in Poultry

Location: Poultry Production and Product Safety Research

2023 Annual Report

1.Implement strategies using plant derived, food-grade phytochemical nanoemulsions for reducing Salmonella and Campylobacter in poultry. 1A.Investigate the efficacy of in-water supplementation of phytochemical nanoemulsions in reducing S. Enteritidis and C. jejuni colonization in broiler chickens. 1B.Reduce Salmonella and Campylobacter on chicken carcasses using phytochemical nanoemulsions applied as a post-harvest intervention at critical control points in processing plants. 1C. Determine the quality, shelf-life and consumer acceptability of chicken meat subjected to the aforementioned interventions. 2. Investigate the potential mechanism(s) of action of phytochemical nanoemulsions against pathogen biofilms and determine efficacy for reducing Salmonella and Campylobacter biofilms in poultry processing plants. 2A. Determine the efficacy of phytochemical nanoemulsions as an antimicrobial wash for eradicating mature S. Enteritidis and C. jejuni biofilm formed on common food contact surfaces. 2B. Determine the efficacy of phytochemical nanoemulsions as an antimicrobial wash for inhibiting S. Enteritidis and C. jejuni biofilm formation on common food contact surfaces and their effect on exopolysaccharide (EPS) production, extracellular DNA (eDNA) production, and quorum sensing. 2C. Investigate the potential mechanism(s) of action of phytochemical nanoemulsions against pathogen biofilm by using transcriptomic and proteomic approaches. 3. Develop vaccine strategies that target multiple pathogens (i.e. Salmonella, Campylobacter, Clostridium, E. coli) utilizing novel Electron-beam technology in poultry. 3A. Test to confirm inactivation of foodborne pathogens in cocktail vaccine consisting of multi-serovars of Salmonella or multiple strains of C. jejuni in broiler chickens. 3B. Determine the efficacy of vaccine consisting of multi- serovars of Salmonella or multiple strains-C. jejuni in reducing colonization and shedding of foodborne pathogens in broiler chickens. 3C. Determine the efficacy of a multi-species cocktail vaccine in reducing colonization and shedding of foodborne pathogens Salmonella enterica, and C. jejuni in broiler chickens. 4. Identify key host neurochemical-microbiota-pathogen interactions across the biogeography of the avian gastrointestinal tract to enhance efficacy of phytochemical and vaccine-based strategies in reducing enteric pathogen colonization. 4A. Determine the ability of heat and cold stressors to influence avian susceptibility to enteric colonization of Salmonella and C. jejuni due to neurochemical production in different regions of the intestinal tract. 4B. Determine functional changes in the microbiome of each region of the avian intestinal tract in response to heat or cold stressors in Salmonella and C. jejuni challenged and unchallenged birds. 4C. Determine the ability of heat and cold stressors to influence efficacies of vaccine and phytochemical modalities on avian susceptibility to enteric foodborne pathogen colonization due to neurochemical production in different... 5.Utilize novel electron-beam technology to reduce pathogen prevalence on poultry products. 5A and 5B, see subobj(s) on uploaded document..

Food safety is a major priority for the poultry industry, among the foodborne pathogens transmitted through poultry products, Salmonella spp. and Campylobacter are epidemiologically linked to the consumption of contaminated poultry and account for the majority of confirmed cases of bacterial gastroenteritis in the US. Despite substantial progress, they remain as the most common foodborne pathogens transmitted to humans. Antibiotic growth promoters (AGPs) have been an integral part of poultry production contributing significantly to controlling pathogens, reducing infections/mortality and improved growth rate. Their use has been restricted in poultry production amid growing concerns of microbial antimicrobial resistance (AMR). The goal of this project is to use a multi-hurdle approach to develop safe and effective alternatives to antibiotics for controlling foodborne pathogens in conventional and organic poultry sectors. First, we will investigate the ability of phytochemical nanoemulsions to reduce Salmonella and Campylobacter colonization in the poultry intestinal tract, on poultry carcasses, and on food contact surfaces. Mechanism of action will be determined as well as the effect of phytochemical intervention on carcass quality and consumer acceptability. Second, electron-beam-technology will be used to develop a safe and effective vaccine targeting both Salmonella and Campylobacter in the chicken intestinal tract. Finally, comprehensive neurochemical and microbial mapping of the poultry gut will determine the effect of stress-related neurochemicals on pathogen colonization and efficacy of phytochemical and vaccine interventions. This research will lead to innovative non-antibiotic intervention strategies using plant-derived antimicrobials and novel vaccine strategies for reducing colonization of foodborne pathogens, decreasing contamination of poultry products and enhancing the health and overall welfare of poultry. Approach for New Objective 5: We proposed to utilize an Electron beam to destroy foodborne pathogens and spoilage organisms in poultry meat and poultry meat products. We will determine an E-beam dose to inactivate Salmonella serovars and Campylobacter jejuni on artificially inoculated poultry meat and poultry products; confirm the efficacy of E-beam dose in inactivating pathogens on naturally contaminated poultry meat and poultry product; and evaluate the quality, shelf-life, and consumer acceptability of E-Beam irradiated meat. Approach for New Subobjectives 5A and 5B. develop research strategies to inactivate foodborne pathogens, Salmonella and Campylobacter, and spoilage bacteria on poultry meat and poultry meat products. We will utilize electron beam (E-Beam) technology to control foodborne pathogens and spoilage organisms in poultry meat and poultry meat products. Inactivation studies will be performed to reduce the pathogen load and spoilage bacteria in meat and meat products. The poultry meat products will also be analyzed for quality, shelf-life, and consumer acceptability of E-Beam-treated meat. Since implementing multiple steps from farm to fork maximizes risk reduction for...See attached for the complete approach.

Progress Report
Progress was made on Objectives 1-4 and the all the proposed subobjectives, all of which fall under National Program 108. Under Objective 1, a study evaluating the efficacy of phytochemical treatments, Trans-cinnamaldehyde (TC) and Caprylic acid (CA), in reducing Salmonella and Campylobacter colonization in market-age broiler chickens has been completed, and we are currently analyzing the data for manuscript preparation. In a separate study, the efficacy of phytochemicals, namely, ginger oil and garlic oil, in reducing Salmonella in the poultry processing environment has been evaluated. A combination of both oils reduced Salmonella on chicken skin by up to 99% and prevented Salmonella growth in the simulated scalding tank environment. Investigation into the antibacterial mode-of-action revealed the potential of the select phytochemicals to inhibit the ability of Salmonella to adhere to host cells. These studies were completed and the manuscript was published. Progress was made on Objective 2’s efforts to understand the potential mechanisms of action of phytochemicals against foodborne pathogens. A study was conducted to understand the relationship between Salmonella infection and the cecal microbiota and the host immune system. Both artificial challenge and natural colonization of the chicken intestinal tract with Salmonella Typhimurium (ST) affected cecal microbiota composition and revealed a decrease in the percentage of circulating monocytes at 7 days post-infection while a decrease in thrombocyte and an increase in heterophil percentages were seen at 14 days post-infection. Taken together, these data provide further insight into the role of the cecal microbiota and host immunity in ST colonization. These studies were completed, and the results were published in the journal of ‘Animals’. Under Objective 3, studies developing vaccine strategies utilizing novel Electron-beam technology in poultry are ongoing. Under Sub-objectives 4A and 4B, bioinformatic processing of intestinal neurochemical and microbial metagenomic datasets from studies into the impact of cold stress on pre-harvest broiler chickens was completed. Cold stress was found to significantly determine relationships between stress-related neurochemicals that drive pathogenesis of foodborne pathogens, and specific gut microbial taxa. Multiple studies investigating the impact of heat stress on pre-harvest broiler chickens were completed. Importantly, cold and heat stressors, were found to shape the developmental trajectory of the intestinal neuroendocrine environment and microbiome as the bird aged, thereby suggesting prevention of stress may hold utility to reduce stress-driven susceptibility to pathogen colonization in later life. A manuscript reporting these results is being prepared for submission. Ex-vivo studies are currently underway that leverage these cold and heat stress findings to identify potential modes of action that block stress-neurochemical driven infection of the chicken gut by Salmonella and Campylobacter.

1. The intestinal tract of chickens is home to a diverse population of bacteria that serve as one of the first lines of defense against pathogenic microbes. ARS researchers in Fayetteville, Arkansas, sought to understand changes in intestinal bacterial populations following Salmonella infection. Their results showed a clear change in the intestinal bacterial structure after Salmonella colonization. An inverse relationship between Salmonella and Lactobacillus and Escherichia was observed, as well as an increase in Bacteroides. Additionally, this work provides insight into the timing of the host immune response, with monocytes and thrombocytes acting early following colonization followed by heterophils. This work demonstrates the ability of Salmonella to change intestinal microbiota composition and highlights genera that may be useful as probiotics to fight Salmonella colonization and promote food safety.

2. Heat stress has been identified to cause changes in stress-related neurochemical concentrations in poultry oviduct and egg contents. However, the mechanism(s) by which heat stress in hens increases vertical transmission of Salmonella, a foodborne pathogen, is largely unknown, thereby limiting producers’ ability to reduce stress-driven incidence of foodborne pathogens in egg production. ARS researchers in Fayetteville, Arkansas, have undertaken research which provides poultry researcjers and industry stakeholders the foundation to explore whether egg neurochemical concentrations could be used as both a non-invasive measure of hen’s stress response and plausibly serve as a mechanism partly responsible for the transmission of Salmonella from hen to egg.

3. Annually, ~95 million people around the world are affected with human campylobacteriosis, a disease caused by Campylobacter jejuni, a foodborne pathogen. C. jejuni from poultry meat products is a leading contributor to the spread of this disease. However, C. jejuni is normal flora in the chicken gut and factors that enable colonization of this bacteria were unknown until recently. ARS researchers in Fayetteville, Arkansas, conducted research to identify several potential factors and target compounds that are involved during the colonization of C. jejuni. Identified factors could be used as intervention tools to control C. jejuni in poultry, thus preventing the spread of C. jejuni as a foodborne illness.

Review Publications
Allem, J., Balasubramanian, B., Rankin, K., Shah, T., Donoghue, A.M., Upadhyaya, I., Sartini, B., Luo, Y., Upadhyay, A. 2023. Trans-cinnamaldehyde nanoemulsion wash inactivates Salmonella Enteritidis on shelled eggs without affecting egg color or embryo growth. Poultry Science. 102(4). Article 102523.
Wagle, B., Quach, A., Yeo, S., Assumpcao, A., Jesudhasan, P., Arsi, K., Donoghue, A.M. 2023. A multiomic analysis of chicken serum revealed the modulation of host factors due to Campylobacter jejuni coloniza-tion and in-water supplementation of eugenol nanoemulsion. Animals. 13(4). Article 559.
Lyte, J.M., Daniels, K.M., Lyte, M., Oluwagbenga, E.M., Fraley, G.S. 2023. Catecholamine concentrations in duck eggs are impacted by hen exposure to heat stress. Frontiers in Physiology. 14. Article 1122414.
Robinson, K., Assumpcao, A., Arsi, K., Donoghue, A.M., Jesudhasan, P. 2022. Ability of garlic and ginger oil to reduce Salmonella in post-harvest poultry. Animals. 12(21):2974.
Robinson, K., Assumpcao, A., Arsi, K., Erf, G.F., Donoghue, A.M., Jesudhasan, P. 2022. Effect of Salmonella Typhimurium colonization on microbiome maturation and blood leukocyte populations in broiler chickens. Animals. 12(20):2867.