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United States Department of Agriculture

Agricultural Research Service

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2010 Annual Report

1a.Objectives (from AD-416)
1) Develop and analyze poultry processing methods that utilize electrolyzed water and antimicrobial fatty acids as microbiocides to decrease microbial contamination during poultry processing. 2) Develop innovative processing operations and recommend modifications in existing processing operations to decrease water use in commercial poultry processing. 3) Evaluate the movement of microorganisms from broiler carcasses to processing water and equipment, specifically scalders, eviscerators and chillers.

1b.Approach (from AD-416)
Alternative antimicrobial treatments such as electrolyzed water, salts of fatty acids, peroxyacetic acid, blends of organic acids, chlorine dioxide, monochloramines, cetylpyridinium chloride and ozone will be evaluated for activity against poultry pathogens and spoilage bacteria. Optimum conditions for applying antimicrobials, including concentration and methods (spray or immersion), will be identified. Treatments with the greatest efficacy against poultry pathogens will be tested further during immersion chilling with reduced water volumes or air chilling. Carcass cross-contamination and procedures of preventing cross-contamination will be determined by focusing experiments on three of the processing areas where the transfer of bacteria is greatest (scalding, defeathering and equipment surfaces). Experiments will also evaluate product contact surfaces as a source of cross-contamination. Partitioning experiments will be performed to separate pathogens on the exterior of the bird from those interior. Cross-contamination will be minimized by using antimicrobial treatments tested in subsequent experiments. Research will be transferred to processing and regulatory personnel for implementation into Sanitation Standard Operating Procedures (SSOPs) and Hazard Analysis and Critical Control Point System (HACCP) plans.

3.Progress Report
Chlorine is currently the most popular sanitizer used in commercial processing in the U.S. Despite the use of chlorine, poultry consumption is still a risk factor associated with human foodborne diseases, and chlorine use has been linked with the formation of carcinogens. Alkaline salts of the fatty acid, lauric acid, are being examined as alternatives to chlorine for use as sanitizers. Research was conducted to examine the ability of spray washing whole broiler carcasses with lauric acid (LA)-potassium hydroxide (KOH) solutions to reduce bacterial contamination of carcasses. The efficacy of spray washing in reducing carcass contamination was influenced by the concentration of LA-KOH used to wash the carcasses and the amount of time carcasses were washed with the LA-KOH solution. Other fatty acids that are being examined for use a sanitizers include myristic, capric, caprylic, and caproic acids. All of these fatty acids are generally recognized as safe. The agar diffusion technique was modified for rapid screening of the antibacterial activity of these compounds. Findings indicate that these fatty acids can kill harmful bacteria associated with poultry processing.

Research was also conducted to calculate the probability of selecting different Salmonella serotypes from agar plates containing two or more serotypes isolated from poultry samples. Since it is not possible to visually differentiate between Salmonella serotypes growing on selective Salmonella agar media, the practice of selecting only one colony from plates containing several colonies may not yield representative samples of Salmonella serotypes that have been recovered. Research was conducted using spreadsheet formulas to calculate probabilities of picking different serotypes from the plate. Findings showed that as the number of different serotypes isolated on the plates increases, more colonies must be picked to ensure that all serotypes that were isolated from the samples are selected. Isolating and serotyping Salmonella from poultry samples is an expensive, labor intensive process; therefore, improved methods are required for ensuring that sampling methods accurately reflect the population of microorganisms found in test samples.

Other research was conducted using phylogenetic analysis to characterize Salmonella Enteritidis isolates recovered from commercial poultry processing operations. Although Salmonella Enteritidis has been previously primarily related to outbreaks of human salmonellosis associated with eating contaminated eggs, an increasing number of cases of this illness have been associated to the consumption of contaminated poultry meat. Pulsed-field gel electrophoresis was used to produce genetic fingerprints of Salmonella isolates recovered from broiler carcasses and water. This research showed a diversity in isolates which indicates that Salmonella isolates may originate from several sources in poultry production system.

1. Determination of Effect of Time, Concentration, and Pressure Used to Spray Wash Carcasses with Solutions of Lauric Acid-Potassium Hydroxide on Bacterial Contamination of Carcasses. Microbicidal surfactants, including solutions of lauric acid-potassium hydroxide, are being examined for use as carcass sanitizers during poultry processing. Studies were conducted to examine the effects of time, concentration, and pressure on the number of bacteria recovered from carcasses after spray washing with solutions lauric acid-potassium hydroxide. Findings indicated that the concentration of the lauric acid-potassium hydroxide solution and the time that carcasses were washed in the solution played significant roles in reducing the number of bacteria recovered from washed carcasses. Altering the pressure used to spray wash carcasses did not produce significant changes in the number of bacteria recovered from washed carcasses, however. These findings will be useful in determining optimal methods for applying alkaline salts of fatty acids as alternative sanitizers during poultry processing.

2. Calculation of the Probability of Identifying Different Salmonella Serotypes Isolated from Poultry Samples. It is not possible to distinguish between Salmonella serotypes by examining the morphology of colonies growing on selective agar media. Research was conducted to examine the probability of selecting Salmonella Enteritidis and Salmonella Typhimurium when both serotypes were present on the same agar plate. Spreadsheet formulas were used to calculate binomial and multinomial probabilities of picking each serotype from the plate. Results indicated that when 2 serotypes are present in equal numbers, 6 colonies must be selected from the plate to have a 95% probability of selecting both serotypes, and 11 colonies must be picked to identify all isolates when 3 serotypes are present. If a serotype is outnumbered 10 to 1 by another serotype on a plate, then 32 colonies must be picked to have a 95% probability of selecting the minority serotype. Results indicate that selecting only one colony per agar plate may not give accurate information on which Salmonella serotype has been isolated from test samples. Given the labor and expense of isolating and serotyping Salmonella isolates, improved methods are required for culturing specific serotypes of interest.

3. Utilization of Phylogenetic Analysis of Salmonella Enteritidis to Characterize Isolates Recovered from Poultry Processing. Salmonella Enteritidis is a cause of foodborne illness associated with consumption of contaminated table eggs. Recently, Salmonella Enteritidis has also become more frequently isolated from broiler poultry carcasses however, raising concern that this Salmonella serotype may also become a significant cause of human foodborne illness associated with consumption of poultry products. Understanding the origins and entry of this human pathogen into integrated poultry production is crucial to identifying control points in the farm-to-fork production continuum. Studies were conducted to determine if endemic strains of Salmonella Enteritidis associated with the poultry processing could be identified and if the source of contamination could be detected. Pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic sequence-polymerase chain reaction (REP-PCR) were used to generate genetic fingerprints of Salmonella Enteritidis isolates from broiler carcasses and water samples collected in a commercial broiler chicken processing plant. The relatedness of the Salmonella isolates was also determined. Antibiotic resistance profiles of the strains indicated that all isolates were sensitive to the antimicrobials tested. Phylogenetic analysis revealed genetic diversity inconsistent with the hypothesis that these isolates originate from a single source within this poultry production system. This study provides data that contribute to understanding the epidemiology of Salmonella Enteritidis in broiler poultry production.

4. Determination of the Role of Antibiotic Pretreatment on the Ability of Salmonella to Colonize Laying Hens. Antibiotics provided to laying hens may influence the ability of Salmonella serotypes to survive in the bird. A study was conducted to evaluate the effects of a vancomycin pretreatment on the ability of nalidixic acid-resistant Salmonella Enteritidis, wild type Salmonella Enteritidis, or nalidixic acid-resistant Salmonella Typhimurium to colonize intestinal and reproductive tracts of laying hens. Half of the group of caged hens was treated with vancomycin, then 6 days later, all hens were challenged orally, intravaginally and intracolonally with Salmonella then placed into separate floor pens on new wood shavings. All hens were killed 2 weeks after Salmonella challenge, and samples were aseptically collected from the ceca, spleen, liver/gallbladder, upper and lower reproductive tracts, and ovarian follicles and cultured for Salmonella. Findings indicated that vancomycin treatment did not significantly effect on the level of colonization of hens by nalidixic acid-resistant S. Enteritidis, wild type Salmonella Enteritidis, or nalidixic acid-resistant Salmonella Typhimurium. Results from these studies may provide further understanding of the mechanism of Salmonella colonization of laying hens.

5. Comparison of Salmonella Recovered from Neck Skin and Whole Carcass Sampling after Air Chilling. The United States (U.S.) and the European Union (E.U.) utilize different sampling methods to test broiler carcasses for the presence of Salmonella. In the U.S., the whole carcass is sampled, while in the E.U. only neck skin taken from carcasses is sampled. Research was conducted to determine the prevalence and serotypes of Salmonella recovered following air chilling of broiler carcasses when neck skin sampling and whole carcass sampling were used to recover the bacterium from chilled carcasses. Both neck skins and whole carcasses of air chilled broilers were incubated in non-selective broth media, and then portions of the media were transferred to selective enrichment broths. Aliquots of the selective broth were streaked onto selective Salmonella agar after incubation, and Salmonella-like colonies on the agar plates were identified. Results indicated that whole carcass enrichment and individual neck skin enrichment sampling methodologies were comparable in the detection of Salmonella from air-chilled carcasses. Therefore, neck skin sampling may be used as a reliable method to accurately detect Salmonella on contaminated, processed broiler carcasses.

6. Determination of the Feasibility of Achieving Zero Tolerance for Salmonella on Raw Poultry. The United States (U.S.) and the European Union (E.U.) utilize different methods to sample broiler carcasses for contamination by Salmonella. The U.S. utilizes the whole carcass rinse sampling method while the E.U. uses a 3 carcass composite of neck skin sample. Both methods were found to be relatively insensitive in detecting Salmonella on raw poultry, and some false negative results were produced. Additionally, commercial processors in the U.S. use immersion chilling and a variety of chemical sanitizers while processors in the E.U. use air chilling and no chemicals during processing. The poultry industry in the U.S. focuses on processing to decrease Salmonella prevalence, but in the E.U. live production is the focus of reducing Salmonella prevalence. A guarantee that all raw poultry meat will be Salmonella free is currently impractical and the absence of Salmonella in processing samples may not mean that the pathogen is not present. Findings indicate that all countries should utilize internationally standardized methods and terminology for sampling poultry samples for the prevalence Salmonella.

7. Examination of Salmonella Enteritidis Growth Characteristics Utilizing Different Enrichment Broths Containing Other Salmonella Serotypes and Extraneous Microflora. Salmonella Enteritidis can be difficult to isolate from broiler breeder flock environmental samples. Studies were conducted to evaluate the growth of non-stressed and stressed Salmonella Enteritidis in buffered peptone water, universal pre-enrichment broth, and tetrathionate broth. Salmonella Enteritidis was cultured in these media with other Salmonella and non-Salmonella microflora. Results indicated that no significant difference in Salmonella Enteritidis growth was observed when pure cultures of the pathogen were grown under various conditions. However, Salmonella Enteritidis growth was significantly reduced when cultured in each enrichment broth with other Salmonella, poultry feces, and poultry feather fluff. Further investigations of factors that affect Salmonella Enteritidis growth during cultivation may enable the development of cultivation methods that enhance recovery Salmonella Enteritidis from environmental samples.

8. Determination of Sensitivity and Selectivity of Cultivation Methods on Recovery Salmonella Serotypes from Hatchery Plenum Samples. Salmonella serotypes exhibit different growth characteristics under the same enrichment conditions, and it is difficult to recover some Salmonella serotypes from poultry samples. A study was conducted to evaluate the serotype diversity of Salmonella recovered from feather fluff samples of broiler hatchery houses when various recovery protocols were used. Findings indicated that cultivation methods may preferentially select some Salmonella serotypes over other serotypes. These findings may explain why Salmonella Enteritidis was not detected by any of the cultivation methods used in this study.

9. Effect of Nisin on the Growth of Listeria monocytogenes on Ready-to-Eat Turkey Ham Stored at Four Degrees Celsius for Sixty-Three Days. Listeria monocytogenes is a major cause of human foodborne diseases associated with refrigerated, ready-to-eat meats that are contaminated by this pathogenic bacterium. The present study was conducted to examine the ability of nisin, rosemary, and ethylenenediaminetetraacetic acid (EDTA) to inhibit growth of Listeria on ready-to-eat, diced turkey ham. The turkey hams were cut into pieces and inoculated with Listeria bacteria. Inoculated ham samples were treated with either nisin; EDTA; rosemary; nisin and EDTA; nisin and rosemary; EDTA and rosemary; or nisin, EDTA, and rosemary. Treated samples were vacuum packaged and refrigerated for up to 63 days. Each week, stored samples were analyzed to determine the number of Listeria bacteria, total bacteria, and lactic acid bacteria present. Nisin; nisin and rosemary; nisin and EDTA; and nisin, rosemary, and EDTA were effective in reducing the population of Listeria bacteria and total bacteria on the ham samples during storage. EDTA, rosemary, or EDTA and rosemary were not ineffective in inhibiting growth of Listeria bacteria during storage. Treatments containing nisin and EDTA were also effective in reducing growth of lactic acid bacteria on the ham during storage. Results from this study indicate that nisin alone or in combination with rosemary and/or EDTA can be used to reduce growth of Listeria bacteria in ready-to-eat turkey ham. These treatments may be considered by processors of ready-to-eat meats as intervention methods to reduce the number of cases of human foodborne illness caused by Listeria.

5.Significant Activities that Support Special Target Populations
Hosted 2 graduate students and one visiting scientist from Tuskegee University. The Tuskegee University students conducted CRIS-related research used to satisfy the research requirement for completion of their Master’s Degree.

Review Publications
Hinton Jr, A., Cason Jr, J.A., Buhr, R.J., Liljebjelke, K.A. 2009. Bacteria recovered from whole-carcass rinsates of broiler carcasses washed in a spray cabinet with lauric acid-potassium hydroxide. International Journal of Poultry Science. 8:1022-1027.

Cox Jr, N.A., Richardson, L.J., Cason Jr, J.A., Buhr, R.J., Vizzier-Thaxton, Y., Smith, D.P., Cray, P.J., Romanenghi, C.P., Pereira, L.B., Doyle, M.P. 2010. Comparison of neck skin excision and whole carcass rinse sampling methods for determining Salmonella prevalence and E. coli counts on broiler carcasses before and after immersion chilling. Journal of Food Protection. 73(5):976-980.

Ruiz, A., Williams, S.K., Hinton Jr, A., Rodrick, G., Dyeri, N. 2010. Nisin affects the growth of listeria monocytogenes on ready-to-eat turkey ham stored at four degrees celsius for sixty-three days. Poultry Science. v:89. p. 353-358.

Last Modified: 6/30/2015
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