Location: Poultry Microbiological Safety and Processing Research Unit
2019 Annual Report
Objectives
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.
Approach
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.
Progress Report
ARS researchers in Athens, Georgia, continued to conduct research on methods to reduce colonization of broiler chickens by human, foodborne pathogens. Additional antimicrobial peptides that can be used to kill pathogens associated with pathogens were identified and screened. The minimum inhibitory concentrations of the peptides required to kill the pathogens was determined and toxicity testing for the peptides has been completed. Salmonella proteins were produced, purified, and tested for the ability to serve as vaccines to inoculate broiler chickens against Salmonella. The single proteins produced a strong immune responses against Salmonella in the chickens; however, combinations of multiple-proteins were required to reduce the level of colonization of poultry by Salmonella. Other research has been conducted to explore the microbial flora of the ceca of broiler chickens. EcoPlates™ were used to characterize the cecal bacteria. Results detected significant differences in the bacterial communities of broilers used in the study. This research will provide commercial poultry growers with methods to reduce the colonization of live chickens by Salmonella and Campylobacter.
Accomplishments
1. Cecal bacterial flora of broiler chickens. ARS researchers at Athens, Georgia, conducted studies to examine the microbial diversity of the ceca of broiler chickens. Analysis of cecal microbial diversity may allow us to develop an effective means to control foodborne pathogens. EcoPlates™ from Biolog, Inc. were used to characterize cecal bacterial communities of six-week-old broiler chickens. Findings indicated that the cecal contents from different broilers metabolized the test substrates at different rates thereby showing that the cecal contents of the broilers contained different amounts and types of bacteria. These findings will be useful in future studies designed to show the role the cecal flora of bacteria in the colonization of broilers by Salmonella and Campylobacter.
Review Publications
Yeh, H., Line, J.E., Hinton Jr, A. 2019. Community-level physiological profiling for microbial community function in broiler cecae. Current Microbiology. 76:173-177.
Gao, Y., Zhuang, H., Yeh, H., Bowker, B.C., Zhang, J. 2019. Effect of rosemary extract on microbial growth, pH, color, and lipid oxidation in cold plasma-processed ground chicken patties. Innovative Food Science and Emerging Technologies. 57:1-6. https://doi.org/10.1016/j.ifset.2019.05.007.
