Location: Poultry Microbiological Safety and Processing Research Unit
2020 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
Sub-Objective 2.1: Broilers can carry Campylobacter in their intestines during live-haul transport to the slaughter plant, and feces from Campylobacter positive birds will contaminate the floor surface of transport containers. Such contamination can be transferred to previously negative broilers when transported in unwashed containers. The use of water rinse, 15 seconds of steam, and a combination of water rinse followed by steam treatment were tested for lessening the numbers of coliforms, Escherichia coli and Campylobacter on squares of broiler transport flooring. Floor squares were covered with 1 g of fresh broiler intestinal contents and then subjected to the various treatments. Water rinse followed by steam heat was significantly (p<0.05) more effective than other treatments by lowering the numbers of all bacteria cultured by 99.99%. Although bacteria were not eliminated, it does show potential to lessen Campylobacter contamination in broiler transport containers.
Sub-Objective 2.2: Broiler carcasses are often contaminated with various human enteropathogens, i.e., Salmonella and Campylobacter species. Removal of these and other pathogens in the processing plant has long been focused on the evisceration side of the plant with little or no strategies utilized on the slaughter side. With the help of industry, ARS researchers in Athens, Georgia, have developed an improved wash cabinet for use before the scald tank to reduce the levels of bacteria on the carcass prior to entering the scald tank. In the pilot processing plant, using water at 450 PSI and 132 F has demonstrated a 2-3 log reduction in the numbers of total bacteria on the carcasses. This reduction in the total number of bacteria would reduce the bacterial load in the scald tanks and thus reduce carcass bacterial contamination.
Sub-Objective 2.3: The ability of Campylobacter to grow in primary containers incubated aerobically or anaerobically was examined. Test media was placed in a flask and inoculated with Campylobacter. The flasks were either closed with plug-sealed caps, vented caps, or vented caps covered with Parafilm laboratory film. Flasks were incubated at 37C for 48 h in an aerobic or anaerobic atmosphere. After incubation, the number of cfu/ml of Campylobacter in the media was enumerated, and the concentration of carbon dioxide in the flasks was measured. Results indicated that the greatest increase in the number of Campylobacter was in the plug-sealed flasks or the parafilm-sealed flasks, while the smallest increase was in the vented flasks. The highest concentration of carbon dioxide was in the plugged flasks. Conclusions indicate that the ability of Campylobacter to grow in the primary containers was related to the ability of the containers to retain CO2 produced by the media and the bacteria. Utilization of this medium will allow laboratories to simplify procedures for culturing Campylobacter.
Sub-objective 3.1: Salmonella Enteritidis and Campylobacter coli inoculation and recovery methods were developed for fertile hatching eggs as a potential model to study the vertical transmission (from hen to chick thru the egg) of these pathogens. The ideal inoculum levels for Salmonella is 1,000 and for Campylobacter is 100 cells/egg. The method was successful for the recovery of Salmonella and Campylobacter for both embryos and egg contents that were inoculated prior to incubation and then incubated for 5 days, but further research is needed to confirm if this is evidence of vertical transmission of the pathogen.
Sub-Objective 4.2: Depending on feed/ingredient type, the pH of bacterial pre-enrichment media can decrease during incubation to a pH of 4.0-5.0. These acidic conditions can kill, injure, or affect bacterial biochemical pathways. Most strains of Salmonella produce hydrogen sulfide from thiosulfate and/or sulfide on selective media and, when combined with iron in the selective plating media, produce FeS (a black compound). On Xylose-Lysine-Tergitol-4 medium, typical Salmonella produce a black centered colony, but when acid stressed, they don’t have this typical appearance and go undetected as an atypical Salmonella isolate. Recovery and detection of Salmonella from feed/feed ingredients is challenging and current methods result in numerous false negative results and should be revisited to improve the rate of detection from feed samples. Two selective enrichment broths and two selective plating media were used for Salmonella recovery from naturally contaminated broiler chicken carcasses rinsates. From 11 of the 49 positive carcasses both plating media (Brilliant Green Sulfa and XLT-4) yielded the same Salmonella serotypes. However, on the other 38 positive samples, different Salmonella serotypes were found on the two plating media. Enrichment and plating media appear to influence the Salmonella serotypes recovered.
Accomplishments
1. New camploybacter medium. As part of research to develop intervention strategies to reduce contamination of processed poultry meat by human, bacterial pathogens, ARS researchers in Athens, Georgia, developed a new method to simplify culturing Campylobacter, a pathogenic bacteria in poultry that does not grow well in the presence of oxygen (aerobically). Researchers inserted Campylobacter-inoculated media in a series of flasks to simulate aerobic (vented cap) and anaerobic (plug-sealed cap or vented cap covered with Parafilm) conditions. The flasks were then incubated in either an aerobic or anaerobic atmosphere. Results indicated that the highest numbers of Campylobacter were recovered from both the plugged and vent covered with Parafilm while the highest concentrations of carbon dioxide were found in the plugged flask in either atmosphere. 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 released from the media or produced by the bacteria. Utilization of this method will simplify procedures for culturing Campylobacter since it will not be required to culture the bacteria in secondary containers with artificially produced microaerobic atmospheres.
Review Publications
Cosby, D.E., Cox Jr, N.A., Berrang, M.E., House, S.L., Line, J.E., Frye, J.G., Jackson, C.R., Hinton Jr, A. 2019. Comparison of two commercially available rapid detection methods and a conventional cultural method to detect naturally occurring salmonellae on broiler carcasses. Journal of Food Safety. 39:e12702. https://doi.org/10.1111/jfs.12702.
Cox Jr, N.A., Oladeinde, A.A., Cook, K.L., Zock, G.S., Berrang, M.E., Ritz, C.W., Hinton Jr, A. 2020. Research Note: Evaluation of several inoculation procedures for colonization of day-old broiler chicks with Salmonella Heidelberg. Poultry Science. 99(3):1615-1617. https://doi.org/10.1016/j.psj.2019.10.020.
Cox Jr, N.A., Berrang, M.E., House, S.L., Hinton Jr, A., Line, J.E., Wiggins, L.T. 2020. Detection of multiple naturally occurring Salmonella serotypes from commercial broiler carcasses with conventional methods. Journal of Food Safety. 40:e12761. https://doi.org/10.1111/jfs.12761.
Kumar, S., Sing, M., Cosby, D.E., Cox Jr, N.A., Thippareddi, H. 2019. Efficacy of peroxy acetic acid in reducing Salmonella and Campylobacter spp. populations on chicken breast fillets. Poultry Science. 99:2655–2661. https://doi.org/10.1016/j.psj.2019.12.045.
Murtada, M., Cosby, D.E., Shanmugasundaram, R., Selvaraj, R. 2020. In vivo and in vitro assessment of commercial probiotic and organi acid feed additives in broilers challenged with Campylobacter coli. Journal of Applied Poultry Research. p. 1-12. https://doi.org/10.1016/j.japr.2020.02.001.
Mclendon, B.L., Cox Jr, N.A., Cosby, D.E., Montiel, E.R., Russell, S.M., Hofacre, C.L., Berrang, M.E., Wilson, J.L. 2020. Detection of salmonella in young chicks with cloacal swabs. Advanced Food and Nutritional Sciences. 5:1-6.
Landrum, M.A., Cox Jr, N.A., Wilson, J.L., Berrang, M.E., Gamble, G.R., Harrison, M.A., Fairchild, B.D., Kim, W.O., Hinton Jr, A. 2019. Reduction of campylobacter on poultry thighs using sequential treatments of antimicrobials. Advanced Food and Nutritional Sciences. 4:1-7. https://doi.org/10.21065.
Huestis, D., Dao, A., Diallo, M., Sanogo, Z., Samake, D., Yaro, S.A., Ousman, Y., Linton, Y., Krishna, A., Veru, L., Krajacic, B., Faiman, R., Florio, J., Chapman, J., Reynolds, D., Weetman, D., Mitchell, R., Donnelly, M., Talamas, E., Chamorro, M.L., Strobach, U., Lehmann, T. 2019. Windborne long-distance migration of malaria mosquitoes in the Sahel. Nature. 28(4):6.
