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:
Objective 1. Develop and evaluate current and novel strategies to reduce food-borne pathogens in the poultry production environment. 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 recombinant Campylobacter jejuni flagellar proteins were expressed, purified and confirmed by detection of the His sequence tag and mass spectrometry. Among them, the FliD protein (flagellar capping protein) was further analyzed by an immunoassay. The result shows that this purified recombinant FliD protein reacted strongly to sera from broiler chickens older than four weeks, indicating that this anti-FliD antibody may be prevalent in the poultry population. In addition, C. jejuni isolates without this protein at the distal end of the flagella greatly reduced their motility and abilities of adhesion and invasion in in vitro studies. In addition, a set of chemotactic proteins of C. jejuni were cloned and expressed. These recombinant proteins were tagged with six-His and hemagglutinin (HA) short sequences at amino and carboxyl termini, respectively. Chicken sera reacted to various numbers of recombinant proteins, but all sera reacted to the Cjj0473 protein (annotated as methyl-accepting chemotaxis protein). These antibody screening results provide rationales for further evaluation of the FliD and Cjj0473 proteins as potential vaccine candidates for broiler chickens to improve food safety for poultry. Objective 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). 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. Two bacteriophage lysin genes (PlyCP39O; PlyCP26F) were codon optimized for expression in yeast. Both proteins have been expressed in yeast by collaborators at the ARS-USDA National Center for Agricultural Utilization Research and were demonstrated to species-specifically lyse Clostridium perfringens. These recombinant proteins will be utilized during experimentation to reduce the levels of Clostridium perfringens in the chicken gastrointestinal tract.
Yeh, H., Hiett, K.L., Line, J.E., Oakley, B., Seal, B.S. 2013. Construction, expression, purification and antigenicity of recombinant Campylobacter jejuni flagellar proteins. Microbiological Research. 168(4):192-198.