Location: Poultry Microbiological Safety and Processing Research Unit
2021 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
This is the final report for the project which was bridged by project 6040-32000-078-000D, "Novel Pre-harvest Interventions and Alternatives to Antibiotics to Reduce Foodborne Pathogens," pending completion of research review. Over the life of the project researchers have made significant progress in discovering new approaches that can be used to reduce the colonization of live poultry by human foodborne pathogens. Research has focused on methods to develop antimicrobial peptides (AMP) and novel vaccines to reduce colonization of live poultry by human, foodborne pathogens.
Under Objective 1, foodborne pathogens and indicator microorganisms were isolated and identified from meat samples that had been stored under an atmospheric cold plasma packaging system. The technology was tested and optimized for treatment of chicken breast filets. The bacterial communities associated with the filets were characterized based on their ability to metabolize various test substrates at different rates. Information from these tests showed that meat samples subjected to different treatments contained different amounts and types of bacteria. In other research several AMP that have the potential to kill human foodborne pathogens, such as Campylobacter, have been screened and some of the peptides have been identified. The minimum inhibitory concentrations of the AMP required to kill the bacteria have been determined, and the testing of the toxicity of the antimicrobial peptides has been completed. Work to determine the stability of the peptides to proteases and other naturally occurring enzymes found in food systems has been conducted. These peptides might be used as replacements for antibiotics that are sometimes used to control colonization of live broilers by harmful microorganisms.
Under Objective 2, poultry samples and samples from farm animals have been analyzed for the presence of Campylobacter bacteria. These samples were taken from seven different local farms. Genetic testing of the Campylobacter bacteria has been conducted, and bacterial isolates were frozen for future analysis. Testing was later expanded to include poultry and other animals from three additional farms. Additionally, proteins from Salmonella bacteria taken from the animals have been cloned and identified. Also, large scale production and purification of these proteins has been completed, and sera taken 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 was used in the development of an experimental vaccine that can be used to make chickens resistant to colonization by Salmonella. As the research progressed, seven other proteins with potential for use as vaccines to immunize broilers chickens against Salmonella were tested. During the tests, Salmonella was orally administered to broilers that had been immunized with the test protein 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 the immunized chickens carried Salmonella. These findings indicated that these proteins can be used as vaccines to reduce Salmonella colonization of live poultry. Further experiments showed that although the single proteins produced a strong immune response against Salmonella in the chickens, combinations of multiple proteins were more effective in reducing the level of colonization of poultry by Salmonella. Work is continuing on production and purification of recombinant Salmonella proteins that may be used for the vaccination of broiler chickens against Salmonella. More than 10 purified recombinant proteins have been obtained. Other research has been conducted to explore the microbial diversity of the cecal contents of 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 of the cecal bacterial flora in the colonization of broilers by Salmonella and Campylobacter.
Under Objective 3, Campylobacter antibody was received from a research collaborator. Tests were conducted to determine the antibody’s reactions with several isolates of Campylobacter, Listeria monocytogenes, Clostridium perfringens, E. coli, Salmonella, Staphylococcus aureus, and Arcobacter.
Results of research conducted under this project has produced several findings that can reduce colonization of live broiler chickens by human foodborne pathogens. Therefore, this research might reduce the number of human foodborne illnesses associated with the consumption of chicken meat.
Accomplishments
1. Draft genome sequences of two Campylobacter jejuni strains showing significantly different colonization potentials in chickens. Poultry is a natural reservoir for Campylobacter jejuni, which is capable of colonizing the chicken intestinal tract. Handling raw chicken meat or eating insufficiently cooked chicken is believed to be a significant risk factor for Campylobacter infection in humans. ARS researchers in Athens, Georgia, isolated a robust colonizer, known as A74/C_24-3, and a poor colonizer, known as A74/O_2-2, exhibiting differing colonization levels after fecal-oral passage through chicks using an individual poultry housing challenge model. DNA was extracted and whole-genome sequencing reactions were performed in collaboration with scientists at FDA. The draft genome sequences of the robust and poor chicken colonizing Campylobacter jejuni isolates were submitted and published in Microbiology Resource Announcements. Whole-genome sequence analyses of these isolates will be helpful in facilitating further studies to identify genetic factors used in chicken colonization.
2. Effect of cold plasma treatment on meat patties. ARS researchers in Athens, Georgia, conducted a study on the effects of different antioxidants on quality of meat patties treated with in-package cold plasma. The results show that although the effectiveness varies with the type of antioxidants utilized, antioxidants such as rosemary, pomegranate and pine bark extracts could enhance anti-bacterial inactivation, suppress lipid oxidation and help maintain appearance of cold plasma-treated poultry meat.
Review Publications
Yeh, H., Line, J.E., Hinton Jr, A., Gao, Y., Zhuang, H. 2020. Bacterial community assessed by utilization of single carbon sources in broiler ground meat after treatment with an antioxidant, carnosine, and cold plasma. Journal of Food Protection, 83 (11): 1967–1973. https://doi.org/10.4315/JFP-20-063.
Sung, K., Khajanchi, B.K., Heitt, K.L., Line, J.E., Khan, S. 2020. Draft genome sequences of two campylobacter jejuni strains that show significantly different colonization potentials in chickens. Microbiology Resource Announcements. 9(41). Article e00687-20. https://doi.org/10.1128/MRA.00687-20.
