Page Banner

United States Department of Agriculture

Agricultural Research Service

Related Topics

Research Project: Molecular Approaches for the Characterization of Foodborne Pathogens in Poultry

Location: Poultry Microbiological Safety Research

2013 Annual Report


1a.Objectives (from AD-416):
1. Utilize genomic, proteomic, and phenotype approaches to improve characterization of foodborne pathogens.

1a. Apply several molecular technologies to further characterize genomic variability of C. jejuni isolates.

1b. Optimize phenotype microarray technology for Campylobacter spp.

1c. Perform proteomic comparisons on the 5 genetically diverse C. jejuni isolates employed in aforementioned genomic characterization investigations (Objective 1: Sub-Objective 1a: C) so as to increase our basic knowledge of differential protein expression.

1d. Perform metagenomic/microbial ecology analyses on chicken gastrointestinal material to increase our basic knowledge of the microbial ecology of the broiler chicken gut.

1e. Develop a database on Campylobacter spp., containing molecular, phylogenetic, proteomic, metabalomic, epidemiologic, and metadata information.

2. Develop, refine, and implement improved methods for the cultural recovery of Campylobacter spp, and potential emerging foodborne pathogens, specifically as it meets regulatory and public health needs.

2a. Utilize genomic, phenotypic, and in silico metabolic reconstruction technologies to develop improved media for isolation of C. jejuni.

2b. Compare existing and newly refined media for recovery of C. jejuni and emerging Campylobacter spp. from the poultry farm environment as well as recovery of isolates most likely involved in public exposure.

3. Using a systems approach, compare Campylobacter spp. with varied colonization abilities for the identification of potential genes or proteins involved in colonization, virulence, and gene expression.

3a. Compare C. jejuni isolates that vary in their ability to colonize broiler chickens, for the identification of genes, proteins, and phenotypes involved in colonization.

3b. Compare C. jejuni isolates that vary in their levels of eukaryotic cell (Caco-2) adherence and invasion for the identification of genes, proteins, and phenotypes involved in virulence.


1b.Approach (from AD-416):
Improved understanding of mechanisms used during poultry colonization as well as defining the impact of applied interventions on Campylobacter spp. and associated chicken microflora is a high priority. The approaches of this project are to.
1)further define novel regions in the Campylobacter spp. genome;.
2)define colonization mechanisms by comparing Campylobacter spp. isolates exhibiting varying degrees of colonization in chickens; and.
3)refine media components and methods for culture based recovery of Campylobacter spp. Our approach follows a coordinate driven model incorporating a comprehensive and multidisciplinary (systems biology) strategy that combines pathogen genomics, proteomics, metabolomics, and metagenomics. The overall impact of this research is to obtain information so as to provide scientifically sound guidance to assist the development of intervention strategies against Campylobacter spp. during broiler production. This should positively impact public health, with a concurrent understanding of the mechanisms and the long-term consequences of intervention application.


3.Progress Report:
Utilize genomic, proteomic, and phenotype approaches to improve characterization of foodborne pathogens. PMSRU investigators combined high-throughput sequencing (HTS), quantitative-PCR assays, and network analysis to profile the poultry-associated microbiome and important pathogens at various stages of commercial poultry production from the farm to the consumer. Network analysis focused on the foodborne pathogen Campylobacter and revealed a majority of sequence types with no significant interactions with other taxa, potentially explaining the limited efficacy of previous attempts at competitive exclusion of Campylobacter.

Develop, refine, and implement improved methods for the cultural recovery of Campylobacter spp, and potential emerging foodborne pathogens, specifically as it meets regulatory and public health needs. PMSRU researchers compared traditional selective media/recovery technologies, a non-selective/filtration (Campycheck) methodology, and various molecular methods (PCR and Next Gen Sequencing) for the detection of Campylobacter spp. from poultry samples (embryonic broiler GI tracts, yolk, broilers raised without antibiotics) and poultry associated environmental samples (soil, poultry litter, processing water, retention pond water, stream water, standing water, other agricultural animals, wild birds). Campylobacter-like colonies were culturally recovered (from these unique matrices) most-often using the Campycheck recovery method. The recovered isolates are currently undergoing further characterization. DNA signatures, for several Epsilon Proteobacteria, were detected using a variety of molecular technologies.

Using a systems approach, compare Campylobacter spp. with varied colonization abilities for the identification of potential genes or proteins involved in colonization, virulence, and gene expression. Differential proteome analysis using 2D-LC-MS/MS was performed to quantitatively measure protein expression changes between robust and poor C. jejuni chicken colonizers. Comparison to the UniProt protein databases generated from the genome-sequenced C. jejuni subsp. jejuni 81176 and 81116, approximately 776 unique proteins were quantified. Seventy-one proteins were differentially expressed between the robust and poor colonizers (parameters: 1.4-fold cutoff and p<0.05). The differentially expressed proteins were primarily associated with metabolic pathways, biosynthesis of secondary metabolites, microbial metabolism in diverse environments, ribosome, ABC transporters, bacterial chemotaxis, and metabolism of cofactors and vitamins, etc. Additional pathway and network analysis of these proteins are still ongoing.


4.Accomplishments
1. Analysis of the poultry microbiome – farm to fork. PMSRU investigators combined high-throughput sequencing (HTS), quantitative-PCR assays, and network analysis to profile the poultry-associated microbiome and important pathogens at various stages of commercial poultry production from the farm to the consumer. Network analysis focused on the foodborne pathogen Campylobacter and revealed a majority of sequence types with no significant interactions with other taxa, potentially explaining the limited efficacy of previous attempts at competitive exclusion of Campylobacter.

2. Identification of proteins involved in C. jejuni colonization of broiler chickens. Differential proteome analysis using 2D-LC-MS/MS was performed to quantitatively measure protein expression changes between robust and poor C. jejuni chicken colonizers. Comparison to the UniProt protein databases generated from the genome-sequenced C. jejuni subsp. jejuni 81176 and 81116, approximately 776 unique proteins were quantified. Seventy-one proteins were differentially expressed between the robust and poor colonizers (parameters: 1.4-fold cutoff and p<0.05). The differentially expressed proteins were primarily associated with metabolic pathways, biosynthesis of secondary metabolites, microbial metabolism in diverse environments, ribosome, ABC transporters, bacterial chemotaxis, and metabolism of cofactors and vitamins, etc. The identification of C. jejuni proteins specifically involved in poultry colonization will facilitate the development of targeted intervention strategies to reduce the pathogen during poultry production.

3. Implement improved methods for the cultural recovery of Campylobacter spp, and potential emerging foodborne pathogens. PMSRU researchers compared traditional selective media/recovery technologies, a non-selective/filtration (Campycheck) methodology, and various molecular methods (PCR and Next Gen Sequencing) for the detection of Campylobacter spp. from poultry samples (embryonic broiler GI tracts, yolk, broilers raised without antibiotics) and poultry associated environmental samples (soil, poultry litter, processing water, retention pond water, stream water, standing water, other agricultural animals, wild birds). Campylobacter-like colonies were culturally recovered (from these unique matrices) most-often using the Campycheck recovery method. The recovered isolates are currently undergoing further characterization. Additionally, DNA signatures for several Epsilon Proteobacteria were detected using molecular technologies. The detection of both “traditionally” non-culturable and emerging Campylobacter spp. is critical to advance our knowledge of the epidemiology of Campylobacter spp.

4. Use of in-package ozone generation technology to reduce poultry meat contamination. ARS scientists in Athens, GA optimized a novel in-package ozonation technology to reduce bacterial contamination on chicken breast filets. Significant reductions of natural bacterial flora and surface-applied bacterial pathogens (Campylobacter jejuni) were achieved using this novel technology. Currently, this technology is being expanded to include major food quality (Pseudomonas fluorescens) and additional food safety (Salmonella spp.) microorganisms. This novel technology will provide commercial processors a method to significantly reduce bacterial pathogens and other bacterial flora on packaged breast filets therefore increasing the quality and safety of the final product as it leaves the processing plant.

5. Method optimization for pathogen ecology studies within poultry production. ARS scientists in Athens, GA demonstrated the differential effects of DNA extraction methods on the molecular analysis of general and pathogen communities within fecal and litter samples with a commercial poultry house. Quantitative (qPCR) and qualitative (next generation sequencing) assessments of microbial communities were significantly affected by DNA extraction methods relative to sample type (litter or feces) and sample environment (dry or moist). Results of this study will provide data to researchers regarding the effects of different extraction methods on the molecular analysis of bacterial communities within live poultry house samples, and provide a basis for comparison of different datasets collected using these different DNA extraction technologies.

6. Development and validation of a Campylobacter genus level qPCR assay. ARS scientists in Athens, GA, developed, optimized, and validated a qPCR assay that specifically targets the Campylobacter genus in a variety of environmental samples. The assay was validated against 16 Campylobacter strains covering major and minor species. The specificity and detection limit of this assay against those strains was determined. The recovery accuracy of this assay was determined through the use of pre-defined combinations of different Campylobacter strains using both cultures and spiked environmental samples. The results of this study will provide researchers with a molecular tool, when used in conjunction with species-specific qPCR assays, to validate culture-based anecdotal information related to the distribution of Campylobacter species in different environmental samples, and provide a new tool to track Campylobacter populations within the environment.

7. Temporal study of pathogen ecology in poultry processing. ARS scientists in Athens, GA demonstrated changes in the bacterial quality of scalder tank water during the commercial processing of poultry. Next generation molecular techniques, combined with cultural recovery, were used to characterize and quantify the bacterial pathogens within these samples, and compare the prevalence of these pathogens to the overall bacterial community and physiochemical characteristics of the processing water. Results of this study will provide data to researchers and commercial processors on the importance of understanding the role of microbial ecology of processing water in controlling poultry food safety-related pathogens in the processing operations.


Review Publications
Hiett, K.L., Cox Jr, N.A., Rothrock Jr, M.J. 2013. Polymerase chain reaction detection of naturally occurring Campylobacter in commercial broiler chicken embryos. Poultry Science. 92(4):1134-1137.

Oakley, B., Morales, C., Line, J.E., Berrang, M.E., Meinersmann, R.J., Tillman, G.E., Wise, M.G., Siragusa, G.R., Hiett, K.L., Seal, B.S. 2013. The poultry-associated microbiome: network analysis and characterization along the farm-to-fork continuum. PLoS One. 8(2):e57190.

Last Modified: 8/1/2014
Footer Content Back to Top of Page