Location: Poultry Microbiological Safety and Processing Research Unit
2024 Annual Report
Objectives
1. Evaluate risk factors that enhance survival of Salmonella and Campylobacter across the poultry processing chain and develop targeted solutions.
1.A Characterize the microbiome of commercial poultry processing facilities to understand how bacterial populations in the processing plant affects the microbial population on processed poultry meat. Special emphasis will be placed on associations between Salmonella in the processing environment microbiome and Salmonella contamination of meat processed in the facility.
1.B Determine Salmonella and Campylobacter prevalence and relative proportion of the pathogens in the resident microbial communities on broiler carcasses after defeathering and prior to chilling operations in commercial poultry processing facilities throughout a processing day.
1.C Create a relational Population Genetics (PopGen) Database of the mobile genetic elements (MGEs) and clustered regularly interspaced palindromic repeats (CRISPR) in different populations of Salmonella and Campylobacter and determine patterns that relate to survival in the poultry environment.
1.D Examine the incidence and importance of aerotolerance and resistance to oxidizing antimicrobial processing aids in Campylobacter and Salmonella isolated from commercial broilers.
2. Develop poultry slaughter and processing control strategies to reduce the quantifiable loads of Salmonella and Campylobacter.
2.A Evaluate the efficacy of antimicrobial applications before, during, or after defeathering, or in sequential combinations after immersion chilling to reduce the load of Salmonella and Campylobacter on broiler carcasses.
2.B Evaluate the reduction of fecal and bacterial contamination of broiler carcasses using a prototype spray cabinet to apply water at varying temperatures and water pressures and to apply chemical interventions combined with varying temperatures and pressures.
Approach
The goal of the project is to reduce contamination of poultry meat during processing by (1) Evaluating risk factors that enhance survival of Salmonella and Campylobacter across the poultry processing chain and (2) Developing poultry slaughter and processing control strategies to reduce the quantifiable loads of Salmonella and Campylobacter.
Progress Report
Objective 1.3: Clustered regularly interspaced short palindromic repeats (CRISPRs) are a mechanism used by bacteria to create immunity to foreign DNA. Genomic sequences for 35,756 isolates of Campylobacter from food animals in the US were collected. 23,797 carried antimicrobial resistance genes. The genomes were screened for CRISPR arrays by two different methods and 106,980 spacers were identified in 18,520 isolates with 10,138 being unique. The spacers are the portion of the CRISPR that confers the targeting capability of CRISPR associated enzymes to destroy specific DNA targets. The sequences of the spacers were compared to sequences in 5 databases to identify their targets. 4,599 of the spacers were against phage (viruses that kill bacteria) and targets for 3,542 could not be identified. It is surmised that many of the unidentified targets are for unknown phage that if found could have potential for phage treatment.
Objective 1.4: Some isolates of Campylobacter are more tolerant to exposure to oxygen. 265 isolates of Campylobacter with various tolerances to oxygen were sequenced and subjected to phylogenetic analysis. Isolates were grouped by tolerance level and the pangenome was analyzed for genes unique to each group. A selection of Salmonella isolates (n=16) predicted to be oxidizer tolerant or sensitive based on previous reports and gene content were tested against peracetic acid and three chlorine containing oxidizers under processing relevant conditions. Oxidizer tolerance was found to be variable and unrelated to gene content. Manuscript submitted and is under review.
Objective 2.1: Serotype specific mixtures of Salmonella were tested against peracetic acid (PAA) applied for 15 seconds in serial with a chlorine containing compound (Cetylpyridinium Chloride, Calcium Hypochlorite, and Sodium Hypochlorite). Thirty-two combinations of concentrations were tested for each pair to select the most effective concentrations to test on meat. This research is in preparation for submission for publication.
Objective 2.2: Neutral Buffered Peptone Water (nBPW) does not select for plasmid of Emerging-Specific Infantis (pESI) positive Salmonella. Since 2017, the percentage of Campylobacter coli isolated from chicken processing plants has risen compared to Campylobacter jeuni. This change has corresponded with the FSIS methods change to rinsing final product samples in nBPW instead of BPW. Investigations of selection of Campylobacter coli over Campylobacter jejuni in nBPW is underway, with the first trial completed.
Non-Assisted Cooperatove Agreement 58-6040-3-028, ARS-University of Georgia: Seven sampling visits were performed across three commercial broiler processing plants. At each visit, samples were collected at hot rehang (after defeathering n=25), post chill (water/ice immersion with added antimicrobial, n=25), and at parts. For evaluating parts, 25 samples were collected each from parts at pre- and post- antimicrobial dip. A total of 700 samples were collected. To address development of novel sampling protocols for improved recovery and detection of Salmonella and Campylobacter, carcass and parts rinses (industry gold-standard; n=10 at each sampling location) were compared to the MicroTally mitt. The mitts were evaluated two ways: 1) using a single mitt on a carcass or on a 4lb bag of parts (n=10 at each sampling location); 2) using a single mitt to sample 50 carcasses at hot rehang and post chill (n=5 at each location) and to sample 100 wings both at pre-dip and post-dip. Measurements from each sample included evaluating 1) Salmonella prevalence by qPCR and culture, 2) Salmonella load using a commercial qPCR assay, 3) Salmonella serovar complexity by deep serotyping. ARS collaborators evaluated Campylobacter prevalence and load, along with Enterobacteriaceae and Aerobic bacteria counts from all samples. As expected, Salmonella and Campylobacter prevalence and load was highest at hot rehang. At this location, use of the three sampling methods (carcass rinse, MicroTally mitt, and multi-carcass MicroTally mitt) were comparable. For the three other locations, pathogen prevalence was significantly lower, and statistical evaluation of the three methods is still being conducted. Sequencing libraries have been generated for Salmonella deep serotyping and these are being analyzed.
Accomplishments
1. Campylobacter contamination and recovery from broiler chicken carcasses is consistent throughout the year and is not seasonal. In the southeastern United States, Campylobacter prevalence in process broiler carcasses is unrelated to season or rainfall, but the genetic diversity of isolates was higher in spring and fall than mid-winter and summer. Campylobacter isolation has previously been shown to be related to season, temperature, and rainfall. Campylobacter was isolated from broiler chicken carcasses at slaughter, weekly, for seven years and isolates were subjected to whole genome sequencing. Prevalence was not related to season, temperature, or rainfall. However, the Shannon’s diversity index of isolate sequences was higher in milder months of the year. ARS researchers in Athens, Georgia, showed that in the southeastern United States. there is a high probability of Campylobacter contamination of broilers throughout the year and will continue to be a year-round concern for both processors and consumers.
Review Publications
Osborne, R.C., Harris, C.E., Buhr, R.J., Kiepper, B.H. 2023. Impact of stunning method on blood loss in broilers during exsanguination with two different neck cut methods. Journal of Applied Poultry Research. 31(1). Article 100385. https://doi.org/10.1016/j.japr.2023.100385.
Reina, M.A., Mcconnell, A., Figueroa, J.C., Riggs, M.R., Buhr, R.J., Price, S.B., Macklin, K.S., Bourassa, D.B. 2023. Quantification of Salmonella Infantis transfer from transport drawer flooring to broiler chickens during holding. Poultry Science. 103(2). Article 10377. https://doi.org/10.1016/j.psj.2023.103277.
Reina, M.A., Urrutia, A., Figueroa, J.C., Riggs, M.R., Macklin, K.S., Buhr, R.J., Price, S.B., Bourassa, D.B. 2023. Application of pressurized steam and forced hot air for cleaning broiler transport container flooring. Poultry Science. 103(2). Article 103276. https://doi.org/10.1016/j.psj.2023.103276.
Shakeri, M., Choi, J., Harris, C., Buhr, R.J., Kong, B.C., Zhuang, H., Bowker, B.C. 2024. Reduced ribonucleotide reductase RRM2 subunit expression increases DNA damage and mitochondria dysfunction in woody breast chickens. American Journal of Veterinary Research. 85(4):1-7. https://doi.org/10.2460/ajvr.23.12.0283.