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Development of Probiotics and Quantitative Assay for Salmonella and Campylobacter in Poultry


Salmonella and Campylobacter are commonly detected in a wide range of poultry and egg products and are responsible for the largest number of cases of foodborne diseases and resulting economic losses in the U.S. yearly. To help ensure a safe supply of poultry products for consumers, the goals of this project are to develop two part approaches (bacteriocin-producing LAB and EMA-LAMP assay) using probiotics to control pathogenic flora and rapid assessments to measure the levels of pathogen contamination. This project will initiate an integrated research project aimed at developing sustainable approaches to control pathogens in poultry farms and processing facilities in the Delaware and Mid-Atlantic region and the nation. <P>The specific objectives of this project are to: 1) initiate a study of probiotics and LAMP techniques that would enhance food microbiology research capacity at Delaware State University (DSU); 2) establish collaborative research with scientists at USDA-ARS, poultry industry, and International Institutes; 3) strengthen the new Master's degree Program in Food Science at DSU, and 4) initiate research which will attract additional and external funds to strengthen our research capacity in Salmonella, Campylobacter, and poultry research. This project will provide direct intervention strategies for Salmonella and Campylobacter contamination in poultry through the development of probiotic strains and monitoring methodologies. In addition, this project will provide students a unique opportunity to gain training in Food Safety and Food Microbiology. Above all, the project will establish a working relationship between Food scientist at DSU and those at the USDA-ARS, the University of Maryland Eastern Shore and poultry industry. <P>Potential products from this research are: isolation of bacteriocin producing LABs for probiotic interventions, development of an EMA-LAMP assay for discriminating viable and dead pathogens, and graduates with MS degree. In addition, peer reviewed journal articles and presentations at scientific meetings, and students' thesis generated from this project will serve as important outcomes.

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Non-Technical Summary:<br/>
Foodborne illnesses from poultry products pose a major health concern to consumers and significant economic loses for poultry farmers and processors. Salmonella and Campylobacter contamination during pre- and post-harvest processing have been implicated as the primary causes of outbreaks from poultry and egg. In order to supply safe poultry products to consumers, a multi-directional approach using micro flora control (probiotic) and a rapid assessment assay must be applied to reduce food poisoning cases caused by these pathogens. A probiotic approach to reduce pathogen levels in poultry will involve lactic acid bacteria (LAB) isolated from fecal and cecal contents of healthy broiler chickens, and the Korean traditional LAB fermented food Kimchi. Loop-Mediated Isothermal Amplification (LAMP) assays will be based on DNA amplification with Ethidium Bromide Monoazide (EMA) treatment to discriminate between viable and dead bacteria within 30 min. The overall goals of this project are to: 1) initiate a study of probiotics and LAMP techniques to reduce Salmonella and Campylobacter in poultry; 2) enhance food microbiology research capacity at Delaware State University (DSU); 3) establish collaborative research with scientists at USDA-ARS and International Institutes; 4) strengthen the new Master degree program in Food Science at DSU; 5) provide research opportunities to minority students; 6) initiate research which will attract additional funds from external sources to sustain Salmonella, Campylobacter, and poultry research.
I. Probiotics isolated from poultry and fermented food to reduce level of Salmonella and Campylobacter: Cecal contents and Kimchi will be isolated for LAB strains. Colonies on MRS plate will be randomly selected from plates before being subjected to anti-microbial activity testing. Selected LAB strains will be grown in broth and supernatant will be obtained by filtration. A lawn of Salmonella and Campylobacter will be prepared on agar plates and supernatant will be spotted onto the plates. The plates will be cultivated at 30 C and growth inhibition will be determined. All isolated LABs that are active against Salmonella and Campylobacter will be tested with the following assays: Gram staining, catalase reaction, etc. The LAB strain will be then characterized by their carbohydrate fermentation pattern. The 16S rRNA gene of bacteriocin-producing LAB strains will be sequenced by an automated gene sequencer. From alignment of DNA sequences collected in Genbank database, a phylogenetic tree will be constructed by the MEGA4 program.
II. Development of EMA-LAMP assay to quantify viable Salmonella and Campylobacter from poultry products: Four primers to target distinct regions specific to Salmonella genomic sequences will be used to amplify the invA gene. In addition, four primers specific to the gufA gene of Campylobacter spp., will be used for the LAMP reaction. LAMP will be performed in a reaction mixture containing template DNA, the forward- and backward-inner primers, and the forward- and backward- outer primers. The LAMP reaction will proceed at 65 C for 35 min. For detection and quantification, SYBR Green I will be added to the LAMP reaction to observe the color changes. The intensity of green color will represent the amount of the target pathogen in each reaction tube. Stomacher bags containing whole piece of chicken wing or thigh and peptone buffer will be seeded with Salmonella and Campylobacter. Recovered bacterial solutions will be mixed with the activated carbon preparations to eliminate PCR inhibitors. EMA and the LAMP assay will be used to discriminate between viable and dead Salmonella and Campylobacter. After EMA treatment, cells will be lysed and DNA purified; the resulting templates will be added directly to the LAMP mixture.
III. Data analysis and evaluation plans: the PI and Co-PIs will meet monthly to evaluate research progress and if progress is aligned with outlined project objectives and to make specific plans for continuation of the research. The results collected by scientists and student(s) will be evaluated for accuracy. The PI will summarize results and submit them annually to the funding agency. Project impact will be evaluated based on: 1) ability of isolated LABs to inhibit the growth of Salmonella and Campylobacter, 2) application of developed methodology to assess the safety of poultry products, 3) students' skills and knowledge of microbiology and molecular biological techniques, 4) number and quality of publications and presentations at scientific meetings, and 5) additional funds attracted from Federal and state agencies; and non-governmental sources.

Lee, Jung-Lim; Besong, Samuel; Harter-Dennis, Jeannine ; Gunther, NW
Delaware State University
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