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United States Department of Agriculture

Agricultural Research Service

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Research Project: Pre-Harvest Interventions For Application During Poultry Production To Reduce Food-Borne Bacterial Pathogens

Location: Poultry Microbiological Safety & Processing Research

2012 Annual Report

1a. Objectives (from AD-416):
1. Develop and evaluate current and novel strategies to reduce food-borne pathogens in the poultry production environment. 1.a. Generate and apply chicken egg-yolk antibodies to reduce Salmonella and Campylobacter populations in broiler chickens. 1.b. Enhance the mucus-binding ability of Lactobacillus reuteri and L. salivarius cultures by growing them in mucin-containing media and compare the ability of enhanced and original cultures to reduce colonization of Salmonella and Campylobacter isolates in vivo. 1.c. Investigate the effects of Bacillus subtilis and Lactobacillus reuteri delivered in feed for reduction of Salmonella and Campylobacter colonization in the broiler chicken’s gastrointestinal system. 2. Develop and evaluate potential alternatives to antimicrobials and other interventions in the control of food-borne pathogens (specifically bacteriophage and bacteriophage lytic enzymes, bacteriocins). 2.a. Identify and clone bacteriophage and prophage lytic enzymes using genomics analyses. 2.b. Express lytic enzymes, bacteriocins and holins in yeast.

1b. Approach (from AD-416):
Novel alternatives to traditional antibiotics are urgently needed for food-animal production. The approaches of this project are to 1) evaluate novel biocontrol strategies to reduce bacterial pathogen GI tract colonization of chickens, and 2) identify and characterize the biophysical properties of anti-bacterial peptides and lytic enzymes. Our approach includes application of specific egg-derived immunoglobulin, enhancing probiotic lactobacilli and determining synergism of probiotic treatments. This will be accompanied by isolation and assay of enzymes capable of lysing food-borne pathogens. The lytic enzymes and previously described bacteriocins will also be cloned for enhanced expression in yeast which can be readily incorporated into chicken feed. In vitro bacterial growth inhibition and in vivo chicken trials will be used to determine practical intervention approaches applicable to the poultry industry. By providing novel alternatives to antibiotic usage in poultry, the overall impact of this research will be a reduction in bacterial pathogens associated with chickens.

3. Progress Report:
Campylobacter jejuni is the leading cause of human food-borne disease associated with poultry. Campylobacter proteins involved with bacterial colonization or motility have the potential for use as antigens for vaccination to reduce C. jejuni in the chicken gastrointestinal (GI) tract. Consequently, bioinformatic searches were performed to select genes encoding proteins potentially involved in colonization and motility from the C. jejuni genome. Twelve C. jejuni flagellar proteins (involved in bacterial movement) have been expressed as recombinants, purified to homogeneity and assayed for biochemical purity. These proteins will be assayed during in ovo vaccine procedures as candidates to reduce C. jejuni in the broiler chicken GI tract. In addition, probiotic bacteria Bacillus subtilis and Lactobacillus reuteri have been developed by an industry partner for use as a feed additive to reduce Campylobacter spp. and Salmonella spp. in the chicken gastrointestinal tract. These bacteria will be utilized in recently designed broiler chicken feeding studies to measure bacterial load in the chicken GI tract and the effect on Campylobacter and Salmonella levels. To further our understanding of changes in the microbial ecology associated with pathogen colonization novel fluoresence in-situ hybridiization (FISH) probes were developed to target Bacillus, Lactobacillus, Salmonella, and Campylobacter. PMSRU researchers have optimized the protocol for utilizing FISH directly with cecal samples and are currently working to optimize hybridization conditions with each of these taxon-specific probes. To rapidly analyze acquired fluorescence images, several high-throughput data analysis pipelines were constructed with scripts written for the programs ImageJ, MatLab, and R. FISH is an essential cultivation-independent tool for microbial ecology that will now allow researchers to evaluate the effects of probiotic treatments on chicken gut microbial ecology. Bacteriocins and bacteriophage lytic proteins can be utilized to inhibit the growth or kill bacteria such as Campylobacter jejuni and Clostridium perfringens, agents that cause human food-borne disease. Synthetic genes optimized for expression in yeast encoding the bacteriocin OR7 and bacteriophage lysin Ply39O have been synthesized by a commercial supplier. The recombinant plasmids encoding the gene for bacteriocin OR7 and Ply39O were transformed into the yeast, Pichia pastoris, and positive clones were identified. Subsequent protein expression was induced by standard procedures and demonstrated that the target proteins were expressed as recombinant proteins. The recombinant proteins will be assayed for the ability to inhibit growth of C. jejuni and C. perfringens in the chicken gastrointestinal system during future feeding trials.

4. Accomplishments
1. Identification and cloning of bacteriophage or prophage lytic enzymes using genomic analyses. Due to an increase in reports of antibiotic resistant bacteria, there has been resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently utilized antibiotics. Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne disease as well as non-food-borne human, animal and poultry diseases. Agricultural Research Service researchers in Athens, Georgia isolated two types of bacteriophage from poultry processing plants, those that had long non-contractile tails, members of the family Siphoviridae, and those with short non-contractile tails, members of the family Podoviridae. Several bacteriophage genes were identified that encoded amidases, lysozyme-endopeptidases, and a zinc carboxypeptidase domain not previously reported in viral genomes, that can potentially digest the cell wall of C. perfringens. Additionally, bioinformatic analysis was utilized to identify an amidase lytic enzyme in the genome of Listeria monocytogenes, the leading cause of bacterial food-borne death in humans. Lytic proteins were cloned and expressed as recombinant proteins, then utilized to kill C. perfringens and L. monocytogenes in the laboratory. Future investigations will examine the ability of these phage lytic proteins to control these pathogens in the chicken gastrointestinal system during poultry production. Reducing populations of pathogens associated with poultry during production will lead to fewer pathogens entering the processing plant and reaching the subsequent consumer and will reduce the risk of human food-borne illness.

Review Publications
Morales, C., Oakley, B., Garrish, J.K., Seal, B.S., Siragusa, G.R., Ard, M.B. 2012. Complete genome sequence of the podoviral bacteriophage CP24R virulent for Clostridium perfringens. Archives of Virology. 157:769-772.

Simmons, M., Morales, C., Oakley, B., Seal, B.S. 2012. Recombinant expression of a putative prophage amidase cloned from the genome of Listeria monocytogenes that lyses the bacterium and its biofilm. Probiotics and Antimicrobial Proteins. 4:1-10.

Volozhantsev, N.V., Oakley, B., Morales, C., Verevkkin, V.V., Bannov, V.A., Popova, A.V., Zhilenkov, E.L., Svetoch, E.A., Garrish, J.K., Schegg, K.M., Woolsey, R., Quilici, D.R., Line, J.E., Hiett, K.L., Siragusa, G.R., Seal, B.S. 2012. Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae. PLoS One. Volume/Page:7/e38283.

Last Modified: 06/23/2017
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