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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Poultry Microbiological Safety and Processing Research Unit » Research » Research Project #439051

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

Location: Poultry Microbiological Safety and Processing Research Unit

2022 Annual Report

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. 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. 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. 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. 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. 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. 3.1. Assess the ability of chemical sanitizers to reduce contamination of inoculated, fertile eggs by Salmonella. 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. 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. 4.1. Evaluate media and growth factors and use the findings to develop new, improved technologies for the isolation and propagation of Campylobacter. 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.

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 complete this project.

Progress Report
ARS researchers in Athens, Georgia, have begun to make significant progress in discovering new approaches to reduce contamination of poultry meat by human foodborne pathogens. Under Objective 4.1, ARS scientists have formulated a new, solid bacterial medium that supports the growth of Campylobacter in petri dishes incubated in aerobic atmospheres. The medium is selective because it allows the growth of Campylobacter while inhibiting the growth of other bacteria, and the medium is differential because the appearance of Campylobacter growing on the medium is different from the appearance of other bacteria growing on the medium. The medium has been named CAMPYAIR, and the use of the new medium could help to reduce the costs, equipment, and technical training required for Campylobacter isolation in research, regulatory, and clinical laboratories. ARS researchers in Athens, Georgia, conducted studies to identify the bacterial composition of dust that had settled on surfaces in chicken houses for Objective 3.2. Scientists examined the bacterial composition of settled dust from 2 broiler houses where chicken flocks had been raised for 6 weeks. Results of experiments showed that the dust was contaminated by several types of Salmonella, aerobic bacteria, coliform bacteria, and E. coli. The Salmonella concentration in the dust was highest between 2 to 6 weeks of growing the broiler chicken flocks in the houses. The study demonstrated that the levels of bacteria in poultry houses may change during growth of poultry and that the dust may serve as a source of Salmonella and other bacteria. ARS researchers in Athens, Georgia, conducted a study to examine the presence of Campylobacter in retail chicken livers under Objective 3.1. Exudates (drippings) and rinsates of the livers were examined for the presence of Campylobacter. Experiments showed that Campylobacter could be isolated from all liver samples examined in the study. When added to growth media, liver exudate was found to be significantly more likely than liver rinses to support detection of Campylobacter. These studies show that improperly prepared chicken liver may serve as a source of foodborne illnesses in humans. ARS researchers in Athens, Georgia, conducted a study to determine the ability of poultry processing sanitizers to reduce contamination of chicken livers by Campylobacter and other bacteria under Objective 2.1. Fresh chicken livers were immersed in cetylpyridinium chloride (CPC) or peracetic acid (PAA), and the number of Campylobacter, Escherichia coli, and other bacteria on the surface of the liver and inside of the liver tissues were determined. Also, changes in the appearance of the livers were monitored. Findings indicated that high concentrations of both sanitizers were required to reduce bacterial contamination of the livers; however, these high sanitizer concentrations produced undesirable changes in the appearance of the treated livers. This study indicated that CPC or PAA should not be utilized in the as treatments to reduce bacterial contamination of chicken livers because of the objectionable changes that the sanitizers produced in the appearance of the livers. ARS researchers in Athens, Georgia, conducted studies to determine the ability of bacterial assays to identify Campylobacter isolated from chicken livers from local retail outlets under Objective 4.2. The researchers also examined the antibiotic resistance of the Campylobacter isolated from the livers. The assay identified two species of Campylobacter, Campylobacter jejuni and Campylobacter coli, that were recovered from the chicken livers. Approximately 37 of the chicken liver isolates were found to be resistant to at least one antibiotic that is used to treat infections humans and other animals. Scientists are now conducting research to determine source on the antibiotic resistance in the Campylobacter isolates. ARS researchers in Athens, Georgia, conducted research to describe a recently emerging gene in bacteria that codes for changes in the bacteria that provides resistance to the antibiotic, colistin under Objective 3.2. Colistin is an important antimicrobial agent that is considered a “drug of last resort” for many bacterial infections in humans. The recently discovered bacterial gene can be found on plasmids that are associated with several foodborne, pathogenic bacteria. Research by the scientists also determined the degree of colistin-resistance that the new gene provides the bacteria and the changes that presence of the gene might produce in the DNA of the bacteria. These findings will provide important information that can be used to understand antimicrobial resistance in bacteria. Also under Objective 3.2, ARS researchers in Athens, Georgia, utilized Salmonella Enteritidis and Campylobacter coli inoculation and recovery methods for hatching eggs as a potential model to study transmission of these pathogens from hen to chick via the egg. The ideal inoculum level for Salmonella and Campylobacter was determined to be 100 – 1000 cells/egg injected into either the yolk or albumen. This method was successful for the recovery of Salmonella and Campylobacter from both embryos and egg contents that were inoculated prior to incubation and then incubated for 15 days. Holding inoculated eggs under refrigeration for 2 days prior to incubation improved recovery from albumen inoculated eggs. Additional research is needed to confirm that this in ovo method can provide evidence of vertical transmission of Campylobacter to the hatched chicks.

1. Reduction of time to detect contamination of broiler meat by Salmonella. ARS researchers in Athens, Georgia, developed a method to reduce the amount of time required to detect contamination of broiler meat by Salmonella. Samples were taken from commercial poultry processing facilities, and a new incubation procedure was used to recover Salmonella in the samples. The new incubation method used a bacterial medium that allows Salmonella to grow during incubation while inhibiting the growth of other bacteria. The new procedure also allows different types of Salmonella to be detected and indicated that the prevalence of one type of Salmonella (Salmonella Infantis) increases during poultry processing operations. Use of this new method suggests that the time required for Salmonella isolation can be reduced without negatively affecting detection of Salmonella.

2. Chemical semicarbazide (SEM) could be formed on chicken meat by poultry processing operations. ARS researchers in Athens, Georgia, conducted studies to determine if the chemical semicarbazide (SEM) could be formed on chicken meat by poultry processing operations. The presence of SEM on poultry meat may be used to indicate that the meat has been treated with an antibiotic that has been banned for use in commercial poultry production. Findings of SEM in poultry processed in the U. S. have been used to prevent export of poultry produced in this country. Experiments were conducted by examining the concentration of SEM on poultry meat samples taken from different points along the processing line. Research findings showed that higher SEM concentrations were found on meat samples taken from later processing operations than from samples taken from earlier processing operations. This research is evidence that the chemical, SEM, can be produced on the meat by processing operations and not be the identifier of a banned antibiotic.

Review Publications
Adhikari, P., Rogiewicz, A., Kim, W.K., Cosby, D.E., Cox Jr, N.A., Slominski, B.A. 2022. Effect of enzyme-modified yeast products on Salmonella Enteritidis colonization in different organs of laying hens. Journal of Applied Poultry Research.
Berrang, M.E., Gamble, G.R., Bowker, B.C., Meinersmann, R.J., Cox Jr, N.A., Knapp, S.W. 2021. Cetylpyridinium chloride and peracetic acid to lessen Campylobacter, Escherichia coli and total aerobic bacterial contamination on chicken liver. Journal of Applied Poultry Research.
Obe, T., Berrang, M.E., Cox Jr, N.A., House, S.L., Shariat, N. 2021. Comparison of selective enrichment and plating media for Salmonella isolation from broiler carcasses. Journal of Food Safety. 41(6):e12928.
Oladeinde, A.A., Abdo, Z., Zwirzitz, B., Woyda, R., Lakin, S.M., Press, M.O., Cook, K.L., Cox Jr, N.A., Thomas, J.C., Looft, T.P., Rothrock Jr, M.J., Zock, G.S., Plumblee Lawrence, J.R., Cudnik, D., Ritz, C. 2022. Litter commensal bacteria can limit the horizontal gene transfer of antimicrobial resistance to Salmonella in chickens. Applied and Environmental Microbiology.88(9).
Pal, A., Bailey, M., Talorico, A.A., Kreheling, J.T., Macklin, K.S., Price, S.B., Buhr, R.J., Bourassa, D.V. 2021. Impact of poultry litter Salmonella levels and moisture on transfer of Salmonella through associated in vitro generated dust. Poultry Science. 100(8):101236.
Rasamsetti, S., Berrang, M.E., Cox Jr, N.A., Shariat, N. 2022. Use of selective pre-enrichment in assessing salmonella presence during broiler processing. Poultry Science.
Weinroth, M.D., Clawson, M.L., Arthur, T.M., Wells, J., Harhay, D.M., Strachan, N., Bono, J.L. 2022. Rates of evolutionary change of resident Escherichia coli O157:H7 differ within the same ecological niche. BMC Genomics. 23. Article 275.
Weinroth, M.D., Thomas, K.M., Doster, E., Vikram, A., Schmidt, J.W., Arthur, T.M., Wheeler, T.L., Parker, J.K., Hanes, A.S., Alekoza, N., Wolfe, C., Metcalf, J.L., Morley, P.S., Belk, K.E. 2022. Resistomes and microbiomes of meat trimmings and colon content from culled cows raised in conventional and organic production systems. Animal Microbiome. 4. Article 21.
Weinroth, M.D., Belk, A.D., Dean, C.J., Noyes, N.R., Dittoe, D.K., Rothrock Jr, M.J., Ricke, S.C., Myer, P.R., Henniger, M.T., Ramirez, G.A., Oakley, B.B., Summers, K.L., Miles, A.M., Ault-Seay, T.B., Yu, Z., Metcalf, J., Wells, J. 2022. Considerations and best practices in animal science 16S ribosomal RNA gene sequencing microbiome studies. Journal of Animal Science. 100:1018.
Pal, A., Jackson, A., Urrutia, A., Macklin, K.S., Price, S.B., Buhr, R.J., Bourassa, D.V. 2022. Bacterial composition of settled dust during growout of broiler chickens. Poultry Science.
Pal, A., Riggs, M.R., Urrutia, A., Osborne, R., Jackson, A., Bailey, M.A., Macklin, K.S., Price, S.B., Buhr, R.J., Bourassa, D.V. 2021. Investigation of the potential of aerosolized salmonella enteritidis on internal organ colonization in broilers between age of D 3 to D 21. Poultry Science.
Berrang, M.E., Cox Jr, N.A., Thompson, T.M., Hinton Jr, A., Yeh, H. 2022. Enrichment and direct plating for detection of campylobacter in chicken liver rinse and exudate. Journal of Food Protection.
Levican, A., Hinton Jr, A. 2022. CAMPYAIR, a new medium for Campylobacter spp. isolation without the need for microaerobic atmosphere. Microorganisms.
Mitchell, T.R., Glenn, A.E., Gold, S.E., Lawrence, K.C., Berrang, M.E., Gamble, G.R., Feldner, P.W., Hawkins, J.A., Miller, C.E., Olson, D.E., Chatterjee, D., Mcdonough, C.M., Pokoo-Aikins, A. 2022. Survey of meat collected from commercial broiler processing plants suggests low levels of semicarbazide can be created during immersion chilling. Journal of Food Protection. 85(5):798-802.
Ramirez, G., Keshri, J., Vahrson, I., Garber, A., Berrang, M.E., Cox Jr, N.A., Seal, B., Samuel, A., Oakley, B. 2022. Cecal microbial hydrogen cycling potential is linked to agriculturally desirable phenotypes in chickens. Frontiers in Microbiology.
Ricker, N., Chalmers, G., Whaler, E., Allen, H.K., Meinersmann, R.J. 2022. Genomic changes within a subset of IncI2 plasmids associated with dissemination of mcr-1 genes and other important AMR determinants. Antibiotics.
Yeh, H., Cox Jr, N.A., Hinton Jr, A., Berrang, M.E., Plumblee Lawrence, J.R., Thompson, T.M. 2021. Prevalence and characterization of quinolone resistance in Campylobacter spp. isolated in chicken livers from retail stores in Georgia, USA. Journal of Food Protection.