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United States Department of Agriculture

Agricultural Research Service

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

Location: Poultry Microbiological Safety Research

2012 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:
Objective 1: Molecular characterization of poultry associated pathogens, coupled with next-generation sequencing, led to the observation that distinct Campylobacter subtypes are associated with specific niches during poultry production, processing, and on the final product. This information allowed for the refined selection of isolates for further characterization (phenotype microarrays and genome sequencing). This data will facilitate the development of specific interventions against Campylobacter along the “farm to fork” continuum, and the development of novel detection tools. Knowledge in these areas should result in reduced carriage of foodborne pathogens in poultry.

Objective 2: In an effort to determine if the use of varied media and recovery conditions introduces a bias on the recovery of Campylobacter subtypes during epidemiologic investigations, three integrated broiler flocks were sampled (production, processing, and final product). Samples were cultured for Campylobacter using four different media coupled with four different atmosphere/temperature combinations. Quantitative analyses demonstrated that recovery of Campylobacter was similar among the four media tested, independent of recovery conditions. Subtype analyses of isolates revealed a bias in subtype recovery relative to sample type, media, incubation temperature, and atmosphere. Next-generation sequencing revealed that Campylobacter represented a small proportion (<0.04%) of nucleotide sequences present in feces. Furthermore, next-generation sequencing revealed that more non-Campylobacter sequences were present upon incubation at 42C relative to 37C. Further characterization of distinct niche-dependent subtypes will facilitate the development of specifically targeted interventions at each integrated stage to reduce and eliminate Campylobacter from poultry.

Objective 3: Dose-response/transmission parameters were monitored for Campylobacter challenge in the chicken. Individual housing was determined as the most efficient experimental design for ascertaining dose-response relationships/estimates of transmission. The individual housing model was employed to determine the infectious dose of C. jejuni isolates previously determined to differentially invade Caco-2 eukaryotic cell lines. Preliminary data revealed that highly invasive isolates were stronger colonizers relative to the less invasive isolates. Further characterization of isolates with different invasiveness will allow for the delineation of colonization factors.


4.Accomplishments
1. Comparative media investigation for the recovery of Campylobacter spp. - a systems approach. A significant contributor to the current knowledge gap on the epidemiology of Campylobacter spp. in poultry is the use of several highly selective, cultivation media with sub-optimal performance. In an effort to determine if the use of varied media and recovery conditions introduces a bias on the recovery of Campylobacter subtypes during epidemiologic investigations, ARS researchers in Athens, Georgia sampled three integrated broiler flocks (production, processing, and final product). Samples were cultured for Campylobacter using four different media coupled with four different atmosphere/temperature combinations. Quantitative cultural analyses demonstrated that recovery of Campylobacter was similar among the four media tested, independent of recovery conditions. Subtype analyses of isolates revealed a bias in subtype recovery relative to sample type, media, incubation temperature, and atmosphere. Next-generation DNA sequence analyses revealed that Campylobacter represented a small proportion (<0.04%) of nucleotide sequences present in feces. Furthermore, next-generation sequencing revealed that more non-Campylobacter sequences were present on plates after incubation at 42C relative to 37C. Further characterization of distinct niche-dependent subtypes will facilitate the development of specifically targeted interventions at each integrated stage to reduce and eliminate Campylobacter from poultry.

2. Analysis of the poultry microbiome – farm to fork. ARS researchers in Athens, Georgia used next-generation DNA sequence analysis to characterize the poultry microbiome throughout a ‘farm-to-fork’ continuum. Investigators determined that a core microbiome, including sequence types most closely related to Clostridium, Campylobacter, and Shigella, was present in all sample types tested. Additionally, fecal samples were determined to contain 2-4-times greater taxonomic richness relative to carcass rinse samples. Interestingly, carcasses sampled 48 hours after processing, revealed the greatest proportion of unique taxa, including Prevotella, Veillonella, Leptrotrichia. Retail products were dominated by Pseudomonas, but were also determined to contain sequence types for 27 additional genera, most of which were encountered in on-farm samples. These data represent the first next-generation DNA sequence based characterization of poultry-associated microbiomes along the farm-to-fork continuum and demonstrate the utility of next-generation DNA sequence analysis for the identification of sources of potential zoonoses.

3. Development and optimization of a rapid and specific antibody biosensor for the detection of Campylobacter spp. from environmental samples. ARS researchers in Athens, Georgia, through a funded cooperative agreement, supplied a panel of target Campylobacter antigens for the development of proprietary, commercial polyclonal and monoclonal antibodies that detect diverse C. jejuni isolates as well as a variety of Campylobacter species. Efficacy screening of over 500 polyclonal antibodies were conducted and subsequently resulted in the incorporation of six monoclonal antibodies into the novel commercial biosensor. The development of rapid and sensitive detection technologies, and the subsequent correlation to molecular and cultural analysis, facilitates regulatory needs of FSIS.

4. Optimization of chicken challenge trials. Dose-response and transmission parameters were monitored for Campylobacter challenge in the chicken. Transmission between co-housed animals was determined to.
1)reduce the apparent value of the infectious dose (ID);.
2)increase the variability between replicates; and.
3)produce a distinctive “all-or-nothing” response. Individual housing was determined to be the most efficient experimental design for ascertaining dose-response relationships and estimates of transmission for C. jejuni. The findings of ARS researchers in Athens, Georgia, suggest that the age-dependence of transmissibility between hosts, rather than their susceptibility to colonization, is the mechanism behind the 'lag-phase' reported in commercial broiler flocks, which are typically Campylobacter free the first 14-21 days of life. The individual housing model was employed to determine the ID of distinct C. jejuni isolates previously determined to differentially invade a eukaryotic cell line (Caco-2). Preliminary data revealed that highly invasive isolates were observed to be strong colonizers relative to the less invasive isolates. Further characterization of isolates with different cell invasiveness potentials will allow for the delineation of colonization factors that will subsequently facilitate the development of interventions.

5. Evaluation of TEMPO technology for rapid automation of the most probable number (MPN) technique. The TEMPO instrument was developed to automate the most-probable-number (MPN) technique and reduce the effort required to estimate bacterial populations. Scientists within the ARS in Athens, Georgia, compared the automated MPN technique with traditional microbiological plating methods and Petrifilm methods for estimating the total viable count of aerobic microorganisms (TVC), total coliforms (TC), and Escherichia coli populations (EC) on freshly processed broiler chicken carcasses (postchill whole carcass rinse samples) and cumulative drip-line samples from a commercial broiler processing facility. When samples below the limit of detection were excluded, 92.1% of the total responses were within a single log difference between the traditional plating or Petrifilm methods and the automated MPN method. These results highlighted the benefits, for FSIS needs of use of the automated TEMPO method for FSIS needs.

6. Development and optimization of a fluorescence in situ hybridization (FISH) technique. In an effort to improve in situ microbial ecology investigations of C. jejuni in poultry, ARS scientists in Athens, Georgia, designed and optimized probes for fluorescence in situ hybridization (FISH) detection. The newly designed FISH probes not only successfully targeted Campylobacter at the genus level, but were also able to discriminate among four closely related isolates on the basis of a single base pair mismatch when used in combination with an unlabeled competitor probe. Additionally, protocols were optimized to perform FISH directly with chicken cecal samples. The optimization of FISH as a cultivation-independent tool for microbial ecology investigations will facilitate future investigations of the interaction of C. jejuni with the gastrointestinal tract of chickens.

7. Next-generation DNA sequence analysis validation of a widely cited Campylobacter specific polymerase chain reaction assay. ARS scientists in Athens, Georgia, tested, using next-generation DNA sequence analysis, the specificity of a widely cited Campylobacter spp. specific polymerase chain reaction (PCR) assay. Additionally, researchers described a method for a direct cell suspension PCR to facilitate sample screening. Pyrosequencing results showed the previously developed assay to be extremely (>99%) sensitive. Additionally, two newly designed broad range bacterial primer sets, that have wide applicability as internal amplification controls, were developed.


Review Publications
Oakley, B., Morales, C., Hiett, K.L., Line, J.E., Seal, B.S. 2012. Application of high throughout sequencing to measure performance of commonly used selective cultivation methods for the food-borne pathogen campylobacter. FEMS Microbiology Ecology. 79(2):327-336.

Oakley, B., Line, J.E., Berrang, M.E., Johnson, J., Buhr, R.J., Cox Jr, N.A., Hiett, K.L., Seal, B.S. 2012. Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase with novel internal amplification controls for rapid and specific detection. Diagnostic Microbiology and Infectious Disease. 72(2):131-138.

Last Modified: 10/25/2014
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