1a. Objectives (from AD-416):
1. Develop and evaluate potential alternatives to antimicrobials and other intervention products and strategies to control and reduce foodborne pathogens in poultry and swine. 1a. Select, chemically synthesize and screen antimicrobial peptides (AMP) for ability to kill Campylobacter spp. in vitro. 1b. Evaluate in vitro and in vivo efficacy of the most active AMP products expressed in yeast or plant vectors and, as necessary, develop encapsulation procedures for enhanced stability, site-directed delivery, efficacy and storage of the protein. 1c. Conduct Campylobacter challenge trials in broiler chickens to determine the ability of oral administration of AMP products to reduce Campylobacter colonization and utilize 16S rRNA sequencing to determine the effect of the peptide products on the overall cecal microbiota. 2. Develop, validate and determine the efficacy of a multi-serotype and multi-subunit cross specific vaccine for use in controlling Campylobacter and Salmonella. 2a. Identify epitopes of the pathogens from peptide microarrays using chicken serum samples from the field. 2b. Construct and express of the epitope containing genes in an Escherichia (E.) coli expression system and purify of the recombinant proteins. 2c. Assay of the immune response in broilers to these recombinant proteins. 2d. Examine populations of bacteria in chicken gastrointestinal tract (GIT) after vaccination to determine the effects of vaccines on the microbiota of the GIT. 3. Employ molecular methods to scientifically guide the development of intervention and mitigation strategies for Campylobacter spp. during poultry production and processing. 3a. Continue Whole Genome Sequence (WGS) comparisons of 50 genetically distinct C. jejuni isolates that vary in their ability to colonize broiler chickens. 3b. Evaluate and refine an atmospheric cold plasma based antimicrobial packaging system (ACP) using genetically diverse C. jejuni isolates, and subsequently perform proteomic analyses to determine the mechanisms associated with die-off. 4. Survey local, pastured-raised, multi-commodity, antimicrobial growth promoter (AGP)-free poultry farms for the presence and variability of common foodborne pathogens as well as to establish changes in the microbial ecology along the “local” farm to fork continuum. 4a. Employ microbiological methods for the recovery of Campylobacter spp. from broiler feces during production, environmental samples, and processing samples. 4b. Perform microbiome analyses on samples to establish changes in the microbial ecology along the “local” farm to fork continuum. 5. Develop, refine, and implement improved methods for the detection and recovery of Campylobacter spp. and other potentially emerging foodborne pathogens, so that the methods meet the needs of associated regulatory agencies. 5.a. Continue collaboration with Pathsensors Inc. for the development of rapid and specific antibody biosensor..." 5.b. Compare traditional selective media/recovery technologies and a non-selective/filtration (Campycheck) methodology..." Please see objective 3 of project plan 070-00D for complete subobjectives for what is now objective 5 of this project.
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) Develop and evaluate antimicrobial peptides (AMP) as potential alternatives to current antibiotics to control and reduce foodborne pathogens in poultry, and 2) Develop, validate and determine the efficacy of a multi-serotype and multi-subunit cross specific vaccine for use in controlling Campylobacter and Salmonella. Specifically, we will select, chemically synthesize, and screen a panel of natural and synthetic AMP for ability to kill Campylobacter spp. in vitro. The genes for expression of the most effective AMP will be coupled to the genes encoding a well-defined bacteriophage receptor binding protein (RBP) to enhance specificity of the AMP for Campylobacters and the AMP-RBP construct will be expressed in a yeast for enhanced production of the protein for evaluation of efficacy. Encapsulation protocols will also be developed for enhanced stability, storage and site-directed delivery of the expressed AMP-RBP product and subsequent Campylobacter colonization challenge trials will be conducted in chicken and swine to evaluate the efficacy of the treatment and determine its overall impact on the gastrointestinal tract (GIT) microbiota. In our second approach (vaccine development) we will identify specific epitopes of the pathogens from peptide microarrays using serum samples from mature commercial chickens. We will then construct and express the epitope containing genes in an Escherichia coli expression system, purify the recombinant proteins, and assay the immune response in broilers to the recombinant proteins. Finally, we will examine the populations of bacteria in the chicken GIT after vaccination to determine the effects of the vaccines on the microbiota. Several knowledge gaps exist regarding the persistence and transmission of Campylobacter during the poultry production/processing continuum. To address these gaps, the proposed research will 1) employ molecular methods to characterize and scientifically guide the development of intervention/mitigation strategies for Campylobacter during poultry production/processing; 2) survey local, pastured-raised, antimicrobial growth promoter-free poultry farms for the presence and variability of common food pathogens; establish changes in the microbial ecology; and establish background levels of antimicrobial resistant pathogens in poultry production devoid of exogenous sources of antimicrobial drugs; and 3) continue to develop and test rapid, specific, and sensitive detection and cultural methods for Campylobacter to assist in meeting the needs of both regulatory agencies and the poultry industry.
3. Progress Report:
ARS researchers at Athens, Georgia, conducted research to develop novel vaccines and antimicrobial proteins (AMP) to reduce contamination of live poultry by human, foodborne pathogens. Additional AMP have been screened and some have been identified that have the potential to kill human foodborne pathogens. Minimum inhibitory concentrations of the AMP have been determined and toxicity testing of the AMP has been completed. Recent progress on this research will be presented at the Bacteriocin and Antimicrobial Peptide International meeting, and a manuscript is in preparation. Proteins from Salmonella bacteria have been cloned and identified. Additionally, large scale production and purification of the proteins has been completed. Sera from chickens of various ages have been tested for reactions with these Salmonella proteins. One of the proteins reacted to about 98% of chicken sera tested, indicating that these chickens had been infected with Salmonella. This protein will be used in the development of a vaccine that can make chickens resistant to Salmonella.
1. Preliminary test of Salmonella recombinant proteins as vaccines in broiler chickens. ARS researchers at Athens, Georgia, evaluated seven recombinant proteins for use as vaccines to immunize broilers chickens agains Salmonella. Salmonella was orally administered to broilers that had been immunized with the test proteins and to broilers that had not been immunized. Samples from the broilers were later examined for the presence of Salmonella. Results showed that three out of four of the unimmunized chickens carried Salmonella, but none of immunized chickens carried Salmonella. These findings indicated that these proteins can be used as vaccines to reduce Salmonella colonization of live poultry. Other experiments are being planned to validate these studies.
Yeh, H., Line, J.E., Hinton Jr, A. 2018. Molecular analysis, biochemical characterization, antimicrobial activity and immunological analysis of proteus mirabilis isolated from broilers. Journal of Food Science. 83(3):770-770. 10.1111/1750-3841.14056.
Yeh, H., Kojima, K., Mobley, J.A. 2018. Epitope mapping of salmonella flagellar hook-associated protein, FlgK, with mass spectrometry-based immuno-capture proteomics using chicken (gallus gallus domesticus] sera. Veterinary Immunology and Immunopathology. 201:20-25. https://doi.org/10.1016/j.vetimm.2018.05.006.