Progress 06/29/11 to 04/25/16
Outputs
Progress Report 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. 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. ARS researchers in Athens, Georgia, implemented a polyphasic, systems based approach to facilitate the characterization of variability observed between Campylobacter spp. The research demonstrated impact through further whole genome sequence analyses of diverse Campylobacter isolates and through the optimization of the Biolog Phenotype Microarray technology for use with Campylobacter spp. Using Phenotype Microarray technology, ARS scientists in Athens, Georgia, demonstrated that temperature affected amino acid utilization in Campylobacter coli. Filling critical data gaps related to variability and biochemical pathways associated with Campylobacter spp. facilitates the development of improved microbiological recovery technologies for the organism. ARS investigators in Athens, Georgia, evaluated management strategies and conducted sampling on both conventional farms as well as farms that produce �all natural� (no AGP use), pastured poultry products. Preliminary data revealed that production and more importantly, processing practices can significantly reduce pathogen levels on retail products. Additionally, preliminary cultural data revealed that Campylobacter spp. was most often recovered from complex environmental samples using a non-selective, filtration-based recovery method. During these studies, method optimization for microbial ecology analysis was performed. As innovative technologies emerge, there is a critical need to evaluate and standardize methods. ARS investigators in Athens, Georgia, demonstrated the method used to extract deoxyribonucleic acid significantly affected downstream results, thus a standardized procedure was optimized and implemented. ARS scientists in Athens, Georgia, were among the first to perform microbial ecology investigations related to broiler poultry production and processing. ARS researchers in Athens, Georgia, were the first to conduct differential proteome analyses to quantitatively measure protein expression changes of distinct Campylobacter jejuni isolates that vary in the ability to colonize broiler chickens. Genes coding for several differentially expressed outer membrane proteins were characterized (similarity, antigenicity, three-dimensional modeling), and assessed for development as potential vaccine candidates. Differential proteome expression analyses were also initiated on Campylobacter jejuni isolates subjected to an atmospheric cold plasma-based antimicrobial packaging system to determine the biological pathways involved in reduction/ elimination post-treatment. The identification of the differential expression of proteins relative to specific variables allowed for scientifically guided, targeted intervention strategies to be pursued. Accomplishments 01 Evaluation 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 and a material transfer agreement with a small, commercial business, evaluated and optimized novel commercial biosensors for the rapid, non-destructive, detection of zoonotic pathogens. The development of rapid, sensitive, specific, and non-destructive detection technologies, with the subsequent correlation to molecular and cultural analyses, facilitates needs of regulatory agencies such as the Food Safety Inspection Service. 02 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. Researchers compared traditional selective media/recovery technologies, a non-selective/filtration (Campycheck) methodology, and various molecular methods (polymerase chain reaction, quantitative polymerase chain reaction, and high throughput sequencing) for the detection of Campylobacter spp. from poultry samples (embryonic broiler gastrointestinal 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. Genomic signatures for several related Epsilon Proteobacteria were detected using a variety of molecular technologies. 03 Use of in-package ozone generation technology to reduce poultry meat contamination. ARS scientists in Athens, Georgia, optimized a novel in- package ozonation technology to reduce Campylobacter spp. 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. 04 Comparative analyses of broiler chickens reared commercially, with and without antibiotic growth promoters. Collaborations with several, small-scale, local farms that produce �all natural� (no antimicrobial growth promoter use), pastured poultry products, were established and are currently undergoing sampling over a three-year investigation. A poly-phasic systems-based approach was employed, and includes the cultural recovery (and enumeration) of several zoonotic pathogens, the evaluation (quantitative polymerase chain reaction and microbiomic high throughput sequence analysis) of general bacterial communities, the physiochemical analysis (pH, electrical conductivity, moisture, total carbon/nitrogen, elements) of associated environmental matrices, and the evaluation of management practices used on each farm (obtained via questionnaire). Additionally, all recovered isolates are being subjected to molecular sub-type analysis and antimicrobial susceptibility testing. Given the significance of food safety issues related to an increased awareness of antimicrobial resistance, investigations, both at the specific pathogen level and at the total microbial community level, will substantially increase current knowledge on the effects of using AGPs in poultry production.
Impacts
(N/A)
Publications
- Rothrock Jr, M.J., Hiett, K.L., Guard, J.Y., Jackson, C.R. 2016. Antibiotic resistance patterns of major zoonotic pathogens from all- natural, antibiotic-free, pasture-raised broiler flocks in the Southeastern United States. Journal of Environmental Quality. 45(2):593- 603.
- Yeh, H., Hiett, K.L., Line, J.E., Jagne, J.F., Lauer, D.C. 2015. Seroprevalence in chickens against campylobacter jejuni flagellar capping protein (FliD) in selected areas of the U.S. Zoonoses and Public Health. 63(4):265-70. doi: 10.1111/zph.12237. epub 2015 Nov 25..
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report 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. 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. Investigators in Athens, Georgia established collaborations to evaluate management strategies and conduct sampling on several local farms that produce �all natural� (no antimicrobial growth promoter use), pastured poultry products. Sampling is currently ongoing and will continue through the 2015 season. A polyphasic, systems based approach is being used to evaluate the presence of zoonotic pathogens (thus food safety) and associated microbiome compositions, throughout production, processing, and in the resulting retail products. Preliminary data revealed that specific processing practices significantly reduce pathogen levels on retail products. Researchers compared traditional selective media/recovery technologies, a non-selective/filtration (Campycheck) methodology, and various molecular methods polymerase chain reaction, quantitative polymerase chain reaction, and high-throughput sequencing for the detection of Campylobacter spp. from poultry samples (embryonic broiler gastrointestinal 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 recovered (from these unique matrices) most-often using the Campycheck recovery method. The recovered isolates are currently undergoing further characterization. Genomic signatures, for several Epsilon Proteobacteria, were detected using a variety of molecular technologies. Differential proteome analyses were performed to quantitatively measure protein expression changes of distinct Campylobacter jejuni isolates that vary in the ability to colonize broiler chickens. Genes coding for several differentially expressed outer membrane proteins were characterized (similarity, antigenicity, three-dimensional modeling), and assessed for development as potential vaccine candidates. Differential proteome expression analysis was also initiated on Campylobacter jejuni isolates subjected to an atmospheric cold plasma-based antimicrobial packaging system to determine the biological pathways involved in reduction/elimination post-treatment. Accomplishments 01 Method optimization for microbiomic studies conducted within broiler chicken production and processing environments. The rapidly emerging field of microbiomics is being applied to investigate various aspects of poultry production and processing. As new technologies emerge, there is a need to evaluate the associated methods to determine which are the most informative for a particular agricultural system; additionally, there is the critical need to standardize methods so that independent studies can be effectively compared. ARS scientists in Athens, Georgia demonstrated the differential effects of deoxyribonucleic acid extraction methods on microbiomic analyses of bacterial communities within fecal and litter samples during commercial poultry production and processing. Microbial communities were significantly affected by nucleic acid extraction methods relative to sample type (litter or feces) and sample environment (dry or moist). This study clearly demonstrates that data generated from the analysis of microbial communities (microbiomics) is significantly affected by the nucleic extraction technique used. Therefore, it is critical that extraction methodologies be standardized for all future, related investigations. 02 Temporal evaluation of bacterial pathogen ecology during poultry processing. Bacterial pathogen contamination of poultry products during processing remains a public health issue for the American consumer. ARS scientists in Athens, Georgia used high-throughput sequence analysis, combined with cultural recovery methods, to assess changes in the bacterial quality of scalder and chiller tank water during the commercial processing of poultry. Cultural results revealed that as the number of broilers processed increased throughout the day, the recovery of a multi-drug resistant Salmonella Heidelberg increased from zero to a total of eleven isolates. These findings highlight a potential emerging poultry food safety concern for the industry that may be more widespread than initially considered. 03 Implement non-standard cultural method for the recovery of Campylobacter spp, and related emerging foodborne pathogens. Despite a multitude of investigations on the epidemiology of Campylobacter spp. during poultry production and processing, identification of the most critical sources of transmission remains elusive. A significant reason may be the use of several, diverse, highly-selective cultivation media during different investigations. ARS scientists in Athens, Georgia compared four traditional selective media/recovery technologies, a non- selective/filtration (Campycheck) methodology, and various molecular methods for the detection of Campylobacter spp. and related organisms from small, multi-commodity, antimicrobial growth promoter-free farms. Campylobacter-like colonies were recovered most-often using the non- selective/filtration recovery method. Initial molecular typing analysis of recovered organisms revealed a bias in subtype recovery relative to media. This data suggests that the use of multiple cultural techniques (selective and non-selective) may be warranted during epidemiologic investigations on Campylobacter and more importantly, for the detection of emerging Campylobacter species. 04 Eradication of Campylobacter spp. using a novel atmospheric cold plasma- based antimicrobial packaging system. ARS scientists in Athens, Georgia optimized a novel in-package ozonation technology that, in vivo, entirely eliminated a library of genetically diverse Campylobacter jejuni and other Campylobacter species. Furthermore, application of this technology to broiler breast filets artificially inoculated with C. jejuni resulted in the complete kill of the organisms tested. This novel technology will provide commercial poultry processors a tool that significantly increases the bacterial quality and safety of packaged breast filets with respect to Campylobacter spp. 05 Development 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 and a material transfer agreement, identified six monoclonal antibodies that were specific and sensitive when tested against a panel of diverse Campylobacter jejuni, other Campylobacter species, and unrelated bacteria. Each of the monoclonal antibodies was individually incorporated into six novel commercial biosensors for the detection of Campylobacter species. Specificity and sensitivity tests are currently in progress to evaluate each biosensor. The development of rapid, sensitive, and specific detection technologies for Campylobacter spp. facilitates needs of poultry processors and regulatory agencies such as the Food Safety Inspection Service.
Impacts
(N/A)
Publications
- Rothrock Jr, M.J., Ingram, K.D., Gamble, J., Guard, J.Y., Cicconi-Hogan, K. M., Hinton Jr, A., Hiett, K.L. 2015. The characterization of Salmonella enterica serovars isolated from the scalder tank water of a commercial poultry processing plant: Recovery of a multi-drug resistant S. Heidelberg. Poultry Science. 94(3):467-472.
- Oakley, B.B., Lillehoj, H.S., Kogut, M.H., Kim, W.K., Maurer, J.J., Pedroso, A., Lee, M.D., Collett, S.R., Johnson, T.J., Cox Jr, N.A. 2014. Mini-review of the chicken gastrointestinal microbiome. FEMS Microbiology Letters. 360(2):100-122. doi: 10.1111/1574-6968.12608.
- Rothrock Jr, M.J., Hiett, K.L., Gamble, J., Caudill, A., Cicconi-Hogan, K., Caproaso, G.J. 2014. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples. Journal of Visualized Experiments.94:e52161, doi: 10. 3791/52161.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report 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. 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. Utilize genomic, proteomic, and phenotype approaches to improve characterization of foodborne pathogens. Investigators in Athens, Georgia established collaborations to evaluate management strategies and conduct sampling on several local farms that produce �all natural� (no antimicrobial growth promoter use), pastured poultry products. Sampling is currently ongoing and will continue through the 2014 season. A polyphasic, systems based approach is being used to evaluate the presence of zoonotic pathogens (thus food safety) and associated microbiome compositions, throughout production, processing, and in the resulting retail products. Preliminary data revealed that production and more importantly, processing practices can significantly reduce pathogen levels on retail products. Additionally, preliminary cultural data revealed that Campylobacter spp. was most often recovered from complex environmental samples using a non-selective recovery method. 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. Researches compared traditional selective media/recovery technologies, a non- selective/filtration (Campycheck) methodology, and various molecular methods polymerase chain reaction, quantitative polymerase chain reaction, and high throughput sequencing for the detection of Campylobacter spp. from poultry samples (embryonic broiler gastrointestinal 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. Genomic signatures, for several Epsilon Proteobacteria, were detected using a variety of molecular technologies. Using a systems approach, compare Campylobacter spp. for the identification of potential genes or proteins involved in colonization, virulence, and survival. Differential proteome analysis was performed to quantitatively measure protein expression changes of Campylobacter jejuni in response to acid shock (survival in the host gastrointestinal tract). A total of 726 unique proteins were quantified. One hundred and sixty-two proteins (22.3% of total proteins identified) were differentially expressed upon acid exposure (parameters: 1.4-fold cutoff and p<0.05). The differentially expressed proteins were primarily associated with metabolic pathways (64 proteins), cellular processes and signaling (26 proteins), information storage and processing (26 proteins), or poorly characterized (13 proteins). Additional pathway and network analysis of these proteins are still ongoing. Accomplishments 01 Comparative analyses of broiler chickens reared commercially, with and without antibiotic growth promoters. Collaborations with several, small-scale, local farms that produce �all natural� (no antimicrobial growth promoter use), pastured poultry products, were established and are currently undergoing sampling over a two-year investigation. A poly- phasic systems-based approach was employed, and includes the cultural recovery (and enumeration) of several zoonotic pathogens, the evaluation (quatitative polymerase chain reaction and microbiomic high throughput sequence analysis) of general bacterial communities, the physiochemical analysis (pH, electrical conductivity, moisture, total carbon nitrogen, elements) of associated environmental matrices, and the evaluation of management practices used on each farm (obtained via questionnaire). Additionally, all recovered isolates are being subjected to molecular sub-type analysis and antimicrobial susceptibility testing. Given the significance of food safety issues related to an increased awareness of antimicrobial resistance, investigations, both at the specific pathogen level and at the total microbial community level, will substantially increase current knowledge on the effects of using antimicrobial growth promoters in poultry production. 02 Identification of proteins involved in Campylobacter jejuni acid shock response. Differential proteome analysis using was performed to quantitatively measure protein expression changes of C. jejuni in response to acid shock (survival in the host gastrointestinal tract). A total of 726 unique proteins were quantified. One hundred and sixty-two proteins (22.3% of total proteins identified) were differentially expressed upon acid exposure (parameters: 1.4-fold cutoff and p<0.05). The differentially expressed proteins were primarily associated with metabolic pathways (64 proteins), cellular processes and signaling (26 proteins), information storage and processing (26 proteins), or poorly characterized (13 proteins). The identification of C. jejuni proteins involved in host survival is currently guiding the development of targeted intervention strategies aimed at the reduction of Campylobacter spp. during poultry production. 03 Development and optimization of a fluorescence in situ hybridization technique. In an effort to improve in situ microbial ecology investigations of Campylobacter jejuni in poultry, scientists in Athens, Georgia, designed and optimized probes for fluorescence in situ hybridization detection. The newly designed 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 fluorescence in situ hybridization directly with chicken cecal samples. The optimization of this technique 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. 04 Implement improved methods for the cultural recovery of Campylobacter spp, and potential emerging foodborne pathogens. Researchers in Athens, Georgia compared traditional selective media/recovery technologies, a non-selective/filtration (Campycheck) methodology, and various molecular methods (polymerase chain reaction and high throughput sequence analysis) for the detection of Campylobacter spp. from poultry samples (embryonic broiler gastrointestinal 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, geonmic signatures for several of the 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. 05 Use of in-package ozone generation technology to reduce poultry meat contamination. Scientists in Athens, Georgia 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. 06 Method optimization for pathogen ecology studies within poultry production. Scientists in Athens, Georgia demonstrated the differential effects of deoxyribo nucleic acid extraction methods on the molecular analysis of general and pathogen communities within fecal and litter samples with a commercial poultry house. Quantitative (quatitative polymerase chain raction) and qualitative (high throughput sequencing) assessments of microbial communities were significantly affected by nucleic acid extraction methods relative to sample type (litter or feces) and sample environment (dry or moist). Results of this study provided 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 nucleic acidextraction technologies. 07 Temporal study of pathogen ecology in poultry processing. Scientists in Athens, Georgia 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. 08 Evaluation 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 and a material transfer agreement, supplied a panel of target Campylobacter antigens for the development of proprietary, commercial polyclonal and monoclonal antibodies that detect diverse Campylobacter 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 the Food Safety Inspection Service.
Impacts
(N/A)
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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. 06 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. 07 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.
Impacts
(N/A)
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.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. 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. Accomplishments 01 Comparative media investigation for the recovery of Campylobacter spp. - systems approach. A significant contributor to the current knowledge ga on the epidemiology of Campylobacter spp. in poultry is the use of sever 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 produ . Samples were cultured for Campylobacter using four different media coupled with four different atmosphere/temperature combinations. Quantitative cultural analyses demonstrated that recovery of Campylobact 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 a eliminate Campylobacter from poultry. 02 Analysis of the poultry microbiome � farm to fork. ARS researchers in Athens, Georgia used next-generation DNA sequence analysis to characteri the poultry microbiome throughout a �farm-to-fork� continuum. Investigators determined that a core microbiome, including sequence type 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 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-for continuum and demonstrate the utility of next-generation DNA sequence analysis for the identification of sources of potential zoonoses. 03 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. 04 Optimization of chicken challenge trials. Dose-response and transmissio 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� respon 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 hos rather than their susceptibility to colonization, is the mechanism behi the 'lag-phase' reported in commercial broiler flocks, which are typical 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. 05 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 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 chick carcasses (postchill whole carcass rinse samples) and cumulative drip-li samples from a commercial broiler processing facility. When samples belo 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 highlight the benefits, for FSIS needs of use of the automated TEMPO method for FS needs. 06 Development and optimization of a fluorescence in situ hybridization (FI technique. In an effort to improve in situ microbial ecology investigations of C. jejuni in poultry, ARS scientists in Athens, Georgi designed and optimized probes for fluorescence in situ hybridization (FI detection. The newly designed FISH probes not only successfully target Campylobacter at the genus level, but were also able to discriminate amo four closely related isolates on the basis of a single base pair mismatc when used in combination with an unlabeled competitor probe. Additionall 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 o the interaction of C. jejuni with the gastrointestinal tract of chickens 07 Next-generation DNA sequence analysis validation of a widely cited Campylobacter specific polymerase chain reaction assay. ARS scientists Athens, Georgia, tested, using next-generation DNA sequence analysis, th specificity of a widely cited Campylobacter spp. specific polymerase cha reaction (PCR) assay. Additionally, researchers described a method for direct cell suspension PCR to facilitate sample screening. Pyrosequenci results showed the previously developed assay to be extremely (>99%) sensitive. Additionally, two newly designed broad range bacterial prime sets, that have wide applicability as internal amplification controls, were developed.
Impacts
(N/A)
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.
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