Research Project: Production and Processing Intervention Strategies for Poultry Associated Foodborne Pathogens

Location: Poultry Microbiological Safety and Processing Research Unit

2021 Annual Report

Objectives
Objective 1. Develop reliable and reproducible challenge models with Salmonella and Campylobacter for use in accurately developing, evaluating, and validating processes for reducing pathogen load using various chemical sanitizers. Objective 2. Develop, evaluate, and validate current and novel chemicals, operational protocols, and sampling methodologies used during poultry production and processing of broilers for the reduction and/or control of foodborne pathogens. Sub-objective 2.1. Assess the ability of commercial and novel chemical sanitizers to reduce or eliminate Salmonella, Campylobacter, and Pseudomonas species from inoculated broiler carcasses and parts. Sub-objective 2.2. Examine the effectiveness of chemical sanitizers applied to carcasses before defeathering or before chilling to reduce contamination by Salmonella and Campylobacter carcasses in postchill carcasses. Sub-objective 2.3. Formulate novel microbicidal surfactants from mixtures of medium-chain fatty acids (MCFA) and organic acids (OA) to be used as sanitizers to significantly reduce microbial contamination during poultry processing. Objective 3. Identify and evaluate risk factors in the production, management, transportation, or processing that impact bird/egg contamination with foodborne pathogens and develop intervention strategies to control pathogens in the absence of antibiotics. Sub-objective 3.1. Assess the ability of chemical sanitizers to reduce contamination of inoculated, fertile eggs by Salmonella. Sub-objective 3.2. Identify and evaluate risk factors in the production, management, transportation, or processing that impact broiler contamination with foodborne pathogens and develop intervention strategies to control pathogens in the absence of antibiotics. Objective 4. Determine the extrinsic factors that impact the survival and attachment of microorganisms including evaluating media and growth factors. Develop and validate new improved technologies to isolate and propagate foodborne pathogens. Sub-objective 4.1. Evaluate media and growth factors and use the findings to develop new, improved technologies for the isolation and propagation of Campylobacter. Sub-objective 4.2. Assess accuracy of current laboratory methods in recovering Salmonella from poultry, animal feeds, and dry environmental samples with fermentable substrates available and development of a more efficient pre-enrichment media.

Approach
Poultry products contaminated by Salmonella and Campylobacter continue to be major sources of human foodborne illnesses. Live poultry are sporadically colonized by these pathogens, and the birds may serve as reservoirs for the bacteria without displaying any signs of illness or declines in performance. Cross contamination of carcasses during processing may spread the bacteria to poultry meat which may cause foodborne infections if the meat is not properly handled. Therefore, the primary goal of our research will be to develop novel interventions that may be used by commercial poultry producers and processors to reduce contamination of poultry by Salmonella, Campylobacter, and indicator microorganisms. Novel chemical sanitizers that may be used during processing to reduce carcass contamination by foodborne pathogens will be formulated or identified. Factors that enhance survival of these pathogens will be identified and used to formulate a novel bacteriological medium that will be utilized in research projects to determine the efficacy of currently available and newly developed interventions. The project outcomes will result in additional control measures that will reduce the levels of Salmonella and Campylobacter in broiler flocks and reduce contamination of processed carcasses by these pathogens. These outcomes will enable the poultry industry to achieve Food Safety Inspection Service (FSIS) performance standard goals and to reduce the number of cases of human foodborne illness associated with contaminated poultry products. Research goals will be achieved by utilizing an interdisciplinary approach that incorporates knowledge and skills of the scientists and other scientists who possess skills and resources required to successfully compete this project.

Progress Report
This is the final report for this project. It was replaced with bridging project 6040-32000-077-000D pending completion of research review. Over the life of the project researchers made significant progress in discovering new approaches that can be used to reduce the contamination of poultry meat by human foodborne pathogens. Under Objective 1, progress was made in evaluating techniques to enhance attachment of bacteria to broiler carcass skin for experimental trials. Calcium alginate suspensions and vegetable oils were examined as carriers used to enhance bacterial attachment when inoculating carcasses with Salmonella. These findings will be useful in conduction research using whole broilers and chicken parts that are artificially contaminated with bacteria. Under Objective 2, progress was made in the evaluation of a low acid processing aid to reduce numbers of Campylobacter on broiler breast skin and thigh skin. Also, the effect of acidic conditions on the injury and death of Salmonella was determined. Additionally, researchers collaborated with the Food Safety Inspection Service (FSIS) to develop a neutralizing solution for use in Salmonella verification testing in commercial poultry processing facilities. The neutralizer is now being utilized in testing in commercial processing facilities. Progress was also made on examining the effect combining chemical interventions to enhance the ability of individual interventions to reduce contamination of chicken meat by harmful bacteria. Findings demonstrated that dipping poultry meat in antimicrobial chemical solutions in a specific, sequential order produced significant reduction in contamination of the meat by Campylobacter. Furthermore, in vitro studies were conducted to examine the antibacterial activity of medium-chained-fatty acids (MCFA) dissolved in amino acid solutions. Disc diffusion assays were conducted with the MCFA-amino acids and indicated that some of the mixtures could inhibit the growth of pathogenic bacteria. Additional progress was made on the evaluation of fluidic nozzles and high-water pressure to remove bacteria from broiler carcasses. Studies indicated that washing the carcasses with water under high pressure could reduce contamination by Campylobacter and other bacteria. Researchers also determined that removal of Salmonella and Campylobacter from broiler carcasses may be improved by the application of chemical interventions using the high pressure, low flow, fluidic nozzles. Studies on methods to reduce contamination of the flooring materials used in broiler transport crates showed that the use of water rinse and steam treatments could reduce the number of bacteria on the flooring material. Results showed that water rinse followed by steam heat was more effective than other treatments in reducing the numbers of bacteria on the flooring material. These findings may be useful in reducing the number of harmful bacteria on processed chicken meat. Under Objective 3, the impact of adding cetylpyridinium chloride (CPC) to broiler drinking water as a preharvest Salmonella intervention during feed withdrawal was evaluated. Experiments showed that the amount of water and feed consumed by the broilers decreased as the amount of CPC added to the water was increased. CPC reduced water consumption but did not decrease Salmonella concentrations in the broilers. Additionally, under Objective 3, sampling commercial broiler flocks, at different times prior to shipping to processing facilities, indicated that cecal sampling cannot predict Salmonella prevalence following feed withdrawal. Also, contamination of the respiratory tracts of the birds with Salmonella, total aerobic bacteria, or Enterobacter bacteria did not increase during feed withdrawal. Also, under Objective 3, studies were conducted that showed recovery of inoculated Salmonella from the shells of hatching eggs was enhanced by the partial removal of the eggshell cuticle, complete removal of albumen and yolk, or by inoculation through the shell. Other studies were conducted on methods to reduce the contamination of broiler litter by Salmonella by adding alum powder to recycled litter and providing broilers pro-biotics in the drinking water. Findings indicated that both methods could reduce the number of Salmonella recovered from the litter and from the ceca of broiler chickens. These finding will be useful in reducing colonization of live birds by harmful bacteria. Under Objective 4, studies were conducted to formulate a novel selective, bacteriological medium for growth of Campylobacter in containers incubated aerobically. Conclusions indicate that the ability of Campylobacter to grow in the primary containers was related to the ability of the containers to retain carbon dioxide produced by the media and the bacteria. Studies were conducted to formulate a new selective, bacteriological medium for growth of Campylobacter in containers incubated aerobically. Research highly indicated that selective antibiotics could allow Campylobacter to grow while inhibiting the growth of other bacteria in the media. Utilization of this medium will allow laboratories to simplify procedures for culturing Campylobacter. Additionally, work was conducted on determining the effect of acidic conditions on the injury and death of Salmonella. Depending on feed/ingredient type, the pH of the pre-enrichment media can decrease during incubation to a pH of 4.0-5.0. These acidic conditions can kill, injure, or affect their biochemical pathways. Enrichment and plating media appear to influence the Salmonella serotypes recovered. Results showed that environmental stress made some Salmonella more susceptible to acidity, but other Salmonella became less susceptible. Progress was also made on the use of Clustered-Regularly-Interspaced-Short-Palindromic-Repeats (CRISPR) technology to detect broiler contamination by Salmonella. CRISPR detected significantly more types of Salmonella on the carcasses. These findings will provide new methods for growing and identifying major pathogens associated with poultry. Overall, progress on this project has produced new findings that may reduce the contamination of processed poultry meat and the colonization of live poultry by human foodborne pathogens; thereby, reducing the number of foodborne illnesses associated with the consumption.

Accomplishments
1. Incubate Campylobacter on solid media under aerobic atmospheres. ARS researchers in Athens, Georgia, submitted and invention report for a system designed to incubate Campylobacter on solid media under aerobic atmospheres. Utilization of this system will eliminate the requirement to generate artificial atmospheres when isolating and growing Campylobacter on solid medium. The system will simplify the procedures for working with a major, foodborne pathogen associated with poultry products.

Review Publications
Leonie, J., Bourassa, D.V., Boyal, R.S., Harris, C.E., Bartenfeld Jossel, L.N., Campbell, A., Anderson, G., Buhr, R.J. 2020. Animal welfare assessment of on-farm euthanasia methods for individual, heavy turkeys . Poultry Science. 100:100812. https://doi.org/10.1016/j.psj.2020.11.001.
Bourassa, D.V., Harris, C.E., Bartenfeld Jossel, L.N., Buhr, R.J. 2020. Assessment of stabilized hydrogen peroxide for use in reducing Campylobacter levels and prevalence on broiler chicken wings. Journal of Food Protection. 84(3):449-455. https://doi.org/10.4315/JFP-20-356.
Melo, E., Mcelreath, J.S., Wilson, J.L., Lara, L.J., Cox Jr, N.A., Jordan, B.L. 2020. Effects of a dry hydrogen peroxide disinfection system used in an egg cooler on hatchability and chick quality. Poultry Science. 99:5487-5490. https://doi.org/10.1016/j.psj.2020.05.050.
Mortada, M., Cosby, D.E., Akerele, G., Ramadan, N., Oxford, J., Shanmugasundaram, R., Ng, T., Selvaraj, R.K. 2021. Characterizing the immune response of chickens to Campylobacter jejuni (Strain A74C). PLoS ONE. 16(3):e0247080. https://doi.org/10.1371/journal.pone.0247080.
Rasamsetti, S., Berrang, M.E., Cox Jr, N.A., Shariat, N. 2021. Selective pre-enrichment method to lessen time needed to recover Salmonella from commercial poultry processing samples. Food Microbiology. 99:e103818. https://doi.org/10.1016/j.fm.2021.103818.
Melo, E.F., Mcelreath, J.S., Wilson, J.L., Lara, L.J., Cox Jr, N.A., Jordan, B.L. 2020. Effects of a dry hydrogen peroxide disinfection system used in an egg cooler on hatchability and chick quality. Poultry Science. 99:5487-5490. https://doi.org/10.1016/j.psj.2020.05.050.
Ramirez, G., Richardson, E., Clark, J., Kishri, J., Dreschler, Y., Berrang, M.E., Meinersmann, R.J., Cox Jr, N.A., Oakley, B. 2020. Broiler chickens and early life programming: Microbiome transplant-induced cecal community dynamics and phenotypic effects. PLoS ONE. 15(11). Article e0242108. https://doi.org/10.1371/journal.pone.0242108.
Boyal, R.S., Buhr, R.J., Harris, C.E., Jacobs, L., Bourassa, D.V. 2020. Equipment and methods for poultry euthanasia by a single operator. Journal of Applied Poultry Research. 29(4):1020-1032. https://doi.org/10.1016/j.japr.2020.09.010.
Tolorico, A.A., Bailey, M.A., Munoz, L.R., Chasteen, K.S., Pal, A., Krehling, J.T., Bourassa, D.V., Buhr, R.J., Macklin, K.S. 2021. The use of roller swabs for salmonella detection in poultry litter. Journal of Applied Poultry Research. 30(3):100163-100169. https://doi.org/10.1016/j.japr.2021.100163.