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





- Buhr, Richard - Jeff
- Meinersmann, Richard - Rick
- 2023
- Publications
- Food Safety, (Animal and Plant Products) (108)

Research Project: Intervention Strategies to Control Salmonella and Campylobacter During Poultry Processing **Location:
                            Poultry Microbiological Safety and Processing Research Unit** 2023 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**
                                

ARS researchers in Athens, Georgia, continued to conduct research on new methods to reduce the contamination of processed poultry meat by human bacterial pathogens.

For Objective 1 the evaluation of risk factors enhancing the Salmonella and Campylobacter survival in the processing chain and the development of targeted solutions required access to commercial farms and processing plants and this access was not available due to the prevalence of highly pathogenic Avian Influenza during the past year. However, the following progress has been made in the following sub-objectives. 

For Sub-objective 1.2, access was obtained to a kill-line in a commercial broiler processing plant. Carcasses were collected and held for 2, 4, 6 hours postmortem before scalding and defeathering the same day and no significant intestinal increase in Campylobacter or Salmonella was found over the 6 hours delay postmortem. However, there were consistently significant increases in the number of pathogens on the carcasses for each subsequent sampling of carcass rinses as the additional carcasses (140 carcasses/minute) from subsequent flocks were scalded and defeathered the 6-hour holding period.

Bacteria in the genus Campylobacter are known to extensively share genes with each other by recombination events (Sub-Objective 1.3). The patterns of gene sharing may show which genes give selective advantage to populations of the bacteria in different hosts. ARS researchers in Athens, Georgia, have obtained from the public database at the National Center for Bioinformatics (NCBI) the genomic sequences of 25756 isolates from chickens, 1392 isolates from turkeys, 2051 isolates from swine, and 7582 isolates from cattle. Very few isolates from ducks are at the NCBI, so DNA from duck isolates obtained previously are being sequenced in the ARS lab. Also, isolates from table-egg layer chickens are also being sequenced as a possible unique population. Comparative studies of gene content, bacteriophage content and barriers to recombination are under way.

Campylobacter causes human infections commonly related to poultry. To determine if Campylobacter strains isolated from poultry plants are aerotolerant (Sub-Objective 1.4), the ability of 48 Campylobacter isolates collected from a chicken processing plant to grow aerobically was determined. From three replications, between 44-75% of the tested isolates grew, albeit poorly, in the presence of ambient air depending on the replication. The potential impact of this work will be information about the characteristics of persistent Campylobacter isolates which could lead to the design of new interventions to reduce Campylobacter contamination.

Raw chicken livers have been identified as a source of Campylobacter and can cause human infections if mishandled (Sub-Objective 1.4). In order to assess the risk of Campylobacter to consumers from liver exudate, naturally occurring Campylobacter were examined in exudate from fresh chicken livers under drying conditions. Fresh chicken liver exudate was dispensed onto sponges and slides and allowed both to dry, and concentrations of total aerobic bacteria (TAB), coliforms, and Campylobacter over seven days were measured. It was found that concentrations of TAB and coliforms remained relatively stable while Campylobacter survived to a maximum of 24 hours, but concentrations were highly dependent on the water activity of the sample. This work shows the potential risk of exposure to Campylobacter to consumers if liver exudate is improperly cleaned and left to dry.

In Objective 2, ARS researchers in Athens, Georgia, continue to work in the processing plant to develop new technology and strategies to reduce the quantifiable loads of Salmonella and Campylobacter. A 20+ year old pilot plant 4-bank picker was evaluated for replacement by a commercial equipment supply company to better defeather today’s larger broilers (3 to 4 Kg). The new picker will have 6-banks of picker discs, spray hot water, and use the modern 2-piece shackle to attain more complete carcass defeathering.

As part of Sub-objective 2.1, ARS researchers in Athens, Georgia, evaluated antimicrobials in vitro for use in future experiments with carcasses. These biocide antimicrobials are currently used in poultry processing to reduce microbial loads, but less is known about the synergistic activities of these chemicals when used in combination. The efficacy of peracetic acid, calcium hypochlorite, and cetylpiridinium chloride was tested against cultures representing four serotypes of Salmonella commonly isolated from poultry. Preliminary concentrations that were tested of these biocides are known to be effective on their own against these bacteria. The data obtained in this in vitro study will be use in vivo to demonstrate a synergistic effect or lack thereof. The potential impact of this work is information for poultry producers on the most effective concentrations of biocide chemicals for reduction of human foodborne pathogens.

Broilers entering the plant covered in fecal material and bacteria continue to reduce the effectiveness of the intervention strategies in the processing plant. ARS researchers from Athens, Georgia, in collaboration with industry partners, develop a wash cabinet designed to reduce fecal and bacterial contamination of carcasses (Sub-objective 2.2). Multiple trials have been conducted with this prototype wash cabinet using pressure increments from 450 to 700 psi (increments of 50 psi) and water temperatures from ambient to 60oC. The combination of 60oC water at 450 psi provides the highest level of bacterial reduction and the removal of fecal material from the carcasses. Additionally, three locations in the processing plant were evaluated using the above determined parameters. The location immediately prior to the scald tank was determined to be the location where the most fecal material and bacteria were removed from the carcasses of broilers entering the processing plant. **Accomplishments**
                                

1. Using 16S rRNA gene sequencing for characterization of poultry-associated microbiomes of the alimentary tract segments. ARS researchers in Athens, Georgia, using 16S rRNA gene sequencing for characterization of poultry-associated microbiomes of the alimentary tract segments (crop to cloaca) and to determine the required minimum number of chickens selected from a flock to adequately capture the true diversity of the microbiome within the flock. The use of cloacal swabs has routinely been used as a non-destructive sample method to represent all other segments of the alimentary tract. Our work only compares alimentary tract locations to cloacal swabs within the same six-week-old broiler chicken. The further the distance from the cloaca the poorer microbiome relativeness to the cloacal swab, with the closest segment, the colon, having the most similar microbiome (although all samples were distinct). When the number of reads assigned to Enterobacteriaceae were specifically compared between cloacal swabs and other alimentary tract segments, only the adjacent colon samples were correlated (P = 0.035, R = 0.70). We concluded that cloacal swabs are not a good approximation of the actual microbiome composition at other alimentary tract segments within an individual chicken or for inferring changes to specific bacterial taxa in other segments. The proventriculus and gizzard required the least number of samples to reach the 1% threshold with 6 birds, the duodenum and jejunum required 7 birds, the crop, ileum, colon, and cloacal swab need 8 and finally, the ceca required 10. There were no families that consistently demonstrated a significant difference between cecal pairs, meaning that variation was more likely driven in the individual bird and not reflective of a biological difference between cecal pairs. These data provide new insights guiding appropriate sample size selection within flocks and add to the consensus data regarding cecal pair similarity and destructive versus non-destructive sampling methods. 2. Neutralizing buffered peptone water (nBPW) does not select for Salmonella Infantis carrying the plasmid known as pESI that has genes that confer multiple antimicrobial resistances. Neutralizing buffered peptone water (nBPW) does not select for Salmonella Infantis carrying the plasmid known as pESI that has genes that confer multiple antimicrobial resistances. S. Infantis has recently become the most common serotype of Salmonella isolated from chicken meat samples. ARS researchers in Athens, Georgia, determined the effect of changes in the FSIS protocol, specifically a change to using nBPW for product rinses and overnight storage rather than buffered peptone water (BPW), the growth rates were measured of pESI positive and negative Salmonella Infantis in nBPW and BPW. Additionally, FSIS methods were simulated for overnight storage of rinses overnight. It was found that nBPW did not provide an advantage to pESI positive Infantis. The impact of this research is information for poultry producers that help explain reasons for the expansion of pESI positive S. Infantis in poultry. (NPL/FSIS requested study). 3. The ability of the feed industry to detect Salmonella in feed and feed ingredients currently depends on the methodology used for pre-enrichment of the samples. The ability of the feed industry to detect Salmonella in feed and feed ingredients currently depends on the methodology used for pre-enrichment of the samples. The two current methods have an inherent weakness due to the low pH produced in the pre-enrichment broths by the background bacteria in the samples. The objective of this study by ARS researchers in Athens, Georgia, was to evaluate the capacity of a newly developed triple buffered peptone broth to recover Salmonella under laboratory conditions. The broth, previously demonstrated to maintain pH at a near neutral level during laboratory test procedures, demonstrated an increased ability to recover Salmonella after incubation when compared to two commonly used pre-enrichment broths. This new broth provides industry scientists with a tool to allow better detection of Salmonella contamination in feed and feed ingredients. 4. Evaluate the role of the eggshell in impeding the transfer of Salmonella into the hatchery. ARS researchers in Athens, Georgia, evaluated the role of the eggshell in impeding the transfer of Salmonella into the hatchery. The outermost protein layer, the eggshell cuticle, as well as the eggshell membranes and albumen impact on Salmonella persistence after holding at room temperature for 1, 6, or 24 hr was examined. After eggshell treatments were applied: mechanically or chemically stripped cuticle or eggshells emptied of egg contents; the eggshells were inoculated with Salmonella, as well as inoculated into the air cell or onto unaltered eggshells (that served as positive control). Stripped of the cuticle eggshell did not impact Salmonella recovery compared to positive control eggs. Eggshells with albumen and yolk removed (prior to eggshell inoculation) resulted in higher Salmonella recovery, indicating that the antimicrobial effect of the cuticle occurs in the presence of albumen through the 24 hr holding and may facilitate the Salmonella inhibition. Salmonella recovery at 24 hr post-inoculation was 31%+ for control eggs compared to eggs with stripped cuticles 35%+, empty egg contents 63%+, or when inoculated into the air cell 63%+. Delaying eggshell Salmonella sampling for 24 hr after inoculation resulted in lower recovery (by 38%) indicating that for flock surveillance, eggshell sampling should be done on the day the egg is laid to optimize recovery of Salmonella. Overall, this study highlights the importance of eggshell cuticle, membranes, and albumen on the integrity of egg contents and the recovery of inoculated Salmonella from eggshells. 5. Potential for sharing genes among Campylobacter populations in chickens and environmental water. In general, bacteria in the same habitat will share genetic material, especially if they are the same species. This can be seen as many genes having identical sequences despite evolutionary distance between the observed individuals. In the Northeastern community of Georgia that were surveyed by ARS researchers in Athens, Georgia, there was virtually no evidence of exchange of DNA between chicken and river isolates of Campylobacter. This could be that there were physiological barriers to the exchange of DNA between these populations of the bacteria such as clustered regularly interspaced palindromic repeats (CRISPRs) and restriction/modification enzyme systems. It was found that the two populations had different specificities of their barrier systems. At this time, it cannot be determined if the differences are the cause or the result of the segregation of the populations of Campylobacter. Further research may show the root cause of the segregation of these populations that can be developed into a barrier of the species from chickens. **Review Publications**
                            *Mcmillan, E.A., Berrang, M.E., Read, Q.D., Ramasetti, S., Richards, A.K., Sharlat, N.W., Frye, J.G. 2022. Buffered peptone water formulation does not influence growth of pESI-positive Salmonella serovar Infantis. Journal of Food Protection. https://doi.org/10.1016/j.jfp.2022.100033.*
                                *Meinersmann, R.J., Berrang, M.E., Shariat, N.W., Richards, A.K., Miller, W.G. 2023. Despite shared geography, Campylobacter isolated from surface water are genetically distinct from Campylobacter isolated from chickens. Microbiology Spectrum. 11(2). Article e04147-22. https://doi.org/10.1128/spectrum.04147-22.*
                                *Harris, C.E., Bartenfeld Jossel, L.N., Bourassa, D.V., Buhr, R.J. 2022. Examination of the eggshell cuticle and membranes on their impact of Salmonella Enteritidis or Typhimurium recovery from inoculated and stored eggs. Journal of Applied Poultry Research. https://doi.org/10.1016/j.japr.2022.100297.*
                                *Cosby, D.E., Berrang, M.E., Hinton Jr, A. 2023. Evaluation of a triple buffered peptone broth for detection of Salmonella in broiler feed. Poultry. 2(1):46-53. https://doi.org/10.3390/poultry2010006.*
                                *Weinroth, M.D., Oakley, B.B., Ramirez, G.A., Reyes, A., Harris, C.E., Buhr, R.J. 2022. 16S rRNA gene-based assessment of common broiler chicken sampling methods: evaluating intra-flock sample size, cecal pair similarity, and cloacal swab similarity to other alimentary tract locations. Frontiers in Physiology. https://doi.org/10.3389/fphys.2022.996654.*


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