Location: Poultry Production and Product Safety Research
2024 Annual Report
Objectives
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..
Approach
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
Under Objective 1, in collaboration with the University of Connecticut, we completed a study investigating the effectiveness of trans-cinnamaldehyde nanoemulsion wash in decreasing the survival of Salmonella Enteritidis on table eggs. Trans-cinnamaldehyde nanoemulsions (TCNE) prepared with emulsifiers Tween 80 (Tw.80) or Gum Arabic and lecithin (GAL) as dip treatments, at 34°C, for reducing S. Enteritidis on shelled eggs. TCNE-Tw.80 or GAL treatments (0.06, 0.12, 0.24, 0.48%) were effective in inactivating S. Enteritidis by at least 2 to 2.5 log cfu/egg as early as 1 min of washing time (P < 0.05). Nanoemulsion wash also inhibited trans-shell migration of S. Enteritidis, as compared to control (P < 0.05). The nanoemulsion wash treatments did not affect shell color (P > 0.05). Results suggest that TCNE could potentially be used as an antimicrobial wash to reduce S. Enteritidis on shelled eggs, further studies investigating the effect of TCNE wash treatments on the organoleptic properties of eggs are underway.
We also conducted another study exploring the impact of nanoemulsion wash containing trans-cinnamaldehyde on the development of chicken embryos in fertilized eggs. Day-old fertilized eggs were subjected to TCNE wash treatments at 34°C for 5 min, followed by 18 d of incubation at 37.7°C. Washing of fertilized eggs with TCNE-Tw.80 or GAL at 0.48% concentration did not significantly alter the egg weight, yolk sac and embryo weight compared to control, and also resulted in 95% fertility (P > 0.05), with 11% and 17% combined early and midterm mortality. Results suggest that TCNE could potentially be used as a natural antimicrobial for fertilized egg sanitation. Further studies in industry settings are warranted.
Under objective 2, we are currently investigating the efficacy of several Generally Regarded as Safe (GRAS) status phytochemicals (eg. thymol, carvacrol, naringenin, rutin, reservatrol, trans-cinnamaldehyde) in inhibiting Salmonella biofilm formation and inactivating mature biofilms on common food contact surfaces. These studies are currently underway.
Under Sub-objectives 3A and 3B, using eBeam technology, eBeam-killed-Salmonella vaccine has been developed by lethally inactivating Salmonella. The eBeam-killed-Salmonella vaccine effectively reduced Salmonella colonization in broiler chickens. The vaccine offers both homologous (protection against the same strain) and heterologous protection (protection against different strains), which is a significant advantage. A manuscript is being prepared to present the findings in a journal.
Under Sub-objectives 4A and 4B, the bioinformatic integration of microbial metagenomic datasets and stress-related intestinal neurochemicals from studies into the effect of heat stress during the pre-harvest stage of broiler chicken production was completed. Heat stress significantly impacted functional pathways of the broiler chicken gut microbiome, as well as specific bacterial taxa, that are known to influence susceptibility of chickens to foodborne pathogen carriage. As increases in stress-related intestinal neurochemicals that affect foodborne pathogen growth were found to co-occur with heat stress driven changes in the microbiome, these findings provide insight into how changes at the gut-level due to stress may predict chicken susceptibility to foodborne pathogen carriage. A manuscript reporting these results is being prepared for journal submission.
Under Sub-objective 4C, the concentrations of the phytochemicals trans-cinnamaldehyde and caprylic acid were determined in regions of the broiler chicken gastrointestinal tract. We have aligned the phytochemical data with neurochemical distribution in the broiler chicken gut and have completed a study investigating the impact of early life cold stress on phytochemical-mediated protection against Salmonella infection. The data from this study is currently being analyzed in preparation for manuscript submission.
Under Sub-objective 5A, ground chicken and ground turkey meats were spiked with a cocktail of 5 strains of Salmonella and 5 Campylobacter jejuni. The meats were electron beam treated with different doses: 0 kGy, 1 kGy, 2 kGy, 3 kGy, and 4 kGy, and results revealed that 2 kGy treatment reduced Salmonella and Campylobacter counts by up to 6 logs and aerobic bacterial counts (total bacteria) were below detection limits in samples treated with 4 kGy. In a separate study, ground chicken and turkey meat were purchased from a local grocery store, exposed to eBeam, and evaluated for meat quality and organoleptic properties. The studies are completed, and the data from this study is currently being analyzed for manuscript preparation.
Accomplishments
1. In-feed inclusion of trans-cinnamaldehyde (TC) and caprylic acid (CA) reduces Salmonella and Campylobacter colonization in market-age broiler chickens. One of the missions of the ARS researchers in Fayetteville, Arkansas, has been to provide the poultry industry with antibiotic alternatives for the control of Campylobacter and Salmonella in conventional and the organic poultry sectors. The use of phytochemicals as antimicrobials and bio-preservatives in food products is one such technology that is safe, effective and environmentally friendly. Our results indicate that plant-based, GRAS (Generally Regarded as Safe) status compounds (TC and CA) effectively prevent Salmonella environmental transmission in broiler chickens. Further, the distribution of TC and CA concentrations in the broiler chicken gastrointestinal tract was determined, thereby providing stakeholders with information that enables precision targeting of the broiler chicken gut with efficacious in-feed phytochemicals.
2. Cold stress post-hatch causes functional shifts in the microbiome that correlate with increased intestinal concentrations of neurochemicals that enhance foodborne pathogen viability and growth. Cold stress during the post-hatch window is known to increase later life susceptibility to Salmonella infection in chickens, it is unknown as to why this observation occurs. ARS researchers in Fayetteville, Arkansas, demonstrated that cold stress exposure during the post-hatch window caused functional shifts in the microbiome that were strongly correlated with increases in intestinal concentrations of neurochemicals that are known to enhance foodborne pathogen viability and growth. These findings provide industry stakeholders and poultry researchers actionable targets to prevent early life stress impact on later life foodborne pathogen colonization.
Review Publications
Gheorghe, C.E., Leigh, S., Tofani, G.S., Bastiaanssen, T.F., Lyte, J.M., Gardellin, E., Martinez-Herrero, S., Goodson, M.S., Kelley-Loughnane, N., Cryan, J.F., Clarke, G. 2024. The microbiota drives diurnal rhythms in tryptophan metabolism in the stressed gut. Cell Reports. 43(4). Article 114079. https://doi.org/10.1016/j.celrep.2024.114079.
Lyte, J.M., Eckenberger, J., Keane, J., Robinson, K., Bacon, T.F., Facchetti, A., Donoghue, A.M., Liyanage, R., Daniels, K.M., Caputi, V., Lyte, M. 2024. Cold stress initiates catecholaminergic and serotonergic responses in the chicken gut that are associated with functional shifts in the microbiome. The ISME Journal: Multidisciplinary Journal of Microbial Ecology. 103(3). Article 103393. https://doi.org/10.1016/j.psj.2023.103393.
Arsi, K., Assumpcao, A., Shyl, D., Donoghue, A.M., Jesudhasan, P. 2024. Draft genome sequences of Campylobacter species isolated from the cecal contents of broiler chickens. Microbiology Resource Announcements. 13(2). Article e00949-23. https://doi.org/10.1128/mra.00949-23.
Lyte, J.M., Arsi, K., Caputi, V., Liyanage, R., Assumpcao, A., Jesudhasan, P., Donoghue, A.M. 2023. Inclusion of trans-cinnamaldehyde and caprylic acid in feed results in detectable concentrations in the chicken gut and reduces foodborne pathogen carriage. Poultry Science. 103(2). Article 103368. https://doi.org/10.1016/j.psj.2023.103368.
Jia, X., Phillips, G., Lyte, J.M. 2023. Complete genome sequence of an avian pathogenic Escherichia coli strain isolated from poultry. Microbiology Resource Announcements. 12(10):e0042423. https://doi.org/10.1128/MRA.00424-23.
Seyoum, M.M., Ashworth, A.J., Owens, P.R., Katuwal, S., Lyte, J.M., Savin, M. 2024. Leaching of antibiotic resistance genes and microbial assemblages following poultry litter applications in karst and non-karst landscapes. Science of the Total Environment. 934. Article 172905. https://doi.org/10.1016/j.scitotenv.2024.172905.