2008 Annual Report
1a.Objectives (from AD-416)
1. Complete the molecular characterization of selected isolates of the pathogens Campylobacter spp., Salmonella spp. and Clostridium perfringens from poultry, utilizing repetitive-sequence PCR. Make a comparison regarding cost and efficiency of identification with other methods of differentiating bacteria, such as, multi-locus sequence analysis or pulsed-field gel.
2. Identify host and pathogen genes important to colonization and/or toxin formation by Campylobacter jejuni and Clostridium perfringens in poultry, and monitor host and pathogen gene expression by RNA microarray analysis. Complete comparative genomic analyses between robust and poor colonizers to identify gene targets that could be disrupted to decrease pathogen presence in the gut environment.
3. Qualitatively and quantitatively identify and compare selected microbial populations in the chicken gastrointestinal and reproductive tracts and in the internal organs of healthy and of diseased birds. Use biophotonics models where appropriate, and develop approaches for processing biofilms to provide for quantitative measurement of bacterial populations in situ.
1b.Approach (from AD-416)
In an effort to further characterize food-borne bacteria, we will (1) perform rep-PCR analysis of Campylobacter spp., Salmonella spp, and Clostridium perfringens isolates, formally recovered from various stages of well-defined poultry production and processing operations; (2) determine if rep-PCR technology can speciate Campylobacter spp.; (3) perform comparisons regarding cost, technical difficulty and efficiency of rep-PCR technology relative to previously established subtyping methods (DNA sequence analysis including multi-locus sequence typing [MLST] and pulse field gel electrophoresis [PFGE]) for each pathogen; and (4) develop an internet accessible database for each poultry associated pathogen assayed by rep-PCR.
In an effort to identify factors responsible for colonization of poultry by Campylobacter jejuni, (1) pathogen gene content and (2) differential gene expression will be monitored by suppressive subtractive hybridization and RNA microarray analysis, respectively. Genes and gene products identified during these experiments will be further characterized and investigated for the development of possible intervention strategies.
In an effort to further our understanding of how environmental changes affect microbial populations associated with poultry production and processing environments we will (1) quantitatively characterize chicken intestinal tract populations in the presence or absence of antimicrobial growth promoters (AGP’s) provided in poultry feed and (2) develop biophotonic methods and techniques to investigate microbial populations in the chicken intestinal tract. Environmental factors (such as AGP in feed) determined to produce changes in microbial populations will be identified and further investigated for the development of possible alternative intervention strategies such as bacteriocins in chicken feed (Stern et al., 2005; 2006).
In an effort to further characterize the role of the fertilized egg in the transmission of Campylobacter spp. in chickens, we will introduce a genetically characterized strain of Campylobacter jejuni (Hiett, et al., 2002), to day-of hatch chicks and test for this particular isolate in the inoculated chickens for a 70 week period. Detection of bacterial pathogens in novel locations in the chicken will allow for the development of more targeted intervention strategies.
In an effort to further our understanding of biofilm formation and persistence in poultry operations, we will (1) develop techniques to label pathogens present in biofilms, especially Listeria monocytogenes, and (2) develop methods for the production of biofilms. Environmental factors contributing to biofilm formation will be identified and further investigated for the development of possible intervention strategies.
All of the progress described herein pertains to Component 1.1 (Pathogens, Toxins, and Chemical Contaminants Pre-harvest) of the 2005-2009 National Program 108 (Food Safety - animal and plant products) Vision-Strategy Document/Action Plan. Emphasis is placed specifically on subcomponents 1.1.3 (Ecology, Host Pathogen and Chemical Contaminants Relationships), and 1.1.4 (Intervention Strategies).
Molecular characterization: We developed a comprehensive repetitive extragenic palindromic-PCR (rep-PCR) fingerprint library to predict serotype for Salmonella isolates for epidemiologic analyses. Semi-automated rep-PCR was evaluated as a method to determine Salmonella serotypes and may be utilized as an alternative to more time-consuming traditional serotyping methods for Salmonella.
The relationship between levels of Campylobacter on contaminated poultry products and human disease showed that implicated broiler product lots (Campylobacter recovered from contaminated chicken flocks that had genetic similarity to Campylobacter isolates recovered from humans with campylobacteriosis) had significantly higher median levels of Campylobacter contamination (~3,600 cells/carcass) than did non-implicated lots (~525 cells/carcass). The differences in mean contamination levels provide a basis for regulatory action beyond that of a presence/absence standard.
Colonization Factors: DNA:DNA (Deoxyribonucleic acid) microarray and suppressive subtractive hybridization analyses were employed to facilitate the identification of factors involved in the colonization of poultry by C. jejuni. Additionally, differential gene expression was monitored using RNA:DNA (ribonucleic acid) microarray analysis and 2-Dimensional Gel Electrophoresis. Several genes and proteins, such as a fibronectin binding protein (cadF) and an outer membrane protein (omp85), were identified as factors potentially involved in colonization of poultry.
Microbial Ecology: Transmission of Salmonella from darkling beetles was investigated. Both adult and larval beetles convey an effective colonization dose to day-of-hatch chicks when gavaged. Salmonella was subsequently shed by the gavaged chicks in feces to colonize non-gavaged pen mate chicks. These results stress the importance of darkling beetle control programs in the overall goal to control.
In an effort to further our understanding of biofilm formation, the development of methods to quantitatively assess pathogens under conditions that mimic food environments was initiated. Bacterial counts from planktonic cultures confirmed that L. monocytogenes was viable throughout the experiments. L. monocytogenes formed biofilms on all of the substrata tested. This is the first use of the crystal violet assay for measurement of bacterial biofilms on stainless steel under these conditions.
Development of Methods for the Production of Biofilms: In an effort to further our understanding of biofilm formation, the development of methods to quantitatively assess pathogens under conditions that mimic food environments was initiated. Stainless steel and glass coupons were incubated in aqueous media containing minimal nutrients and exposed to bacteria under static temperature and humidity conditions. Bacterial counts from planktonic cultures at 24, 48, 72, and 144 h confirmed that L. monocytogenes remained viable throughout the experiments. The cell density log10/ml was 8.01, 8.03, 7.69, and 6.66, respectively. L. monocytogenes formed biofilms on all of the substrata tested. The data will be used to grow stable biofilms of Listeria spp. for further study. This is the first use of the crystal violet assay for measurement of bacterial biofilms on stainless steel under these conditions. The methods tested are applicable to other bacteria and substrata.
Identification of genetic factors involved in Campylobacter spp. colonization of poultry: In an effort to further understand the contamination of poultry with C. jejuni, we sought to identify colonization-associated factors at the genome and proteome level. DNA:DNA microarray hybridization and suppressive subtractive hybridization analyses were employed to facilitate the identification of factors involved in the colonization of poultry by C. jejuni. Additionally, differential gene expression was monitored using RNA:DNA microarray analysis and 2-Dimensional Gel Electrophoresis. Several genes and proteins, such as a fibronectin binding protein (cadF) and an outer membrane protein (omp85), were identified as factors potentially involved in colonization of poultry. Additionally, 98 C. jejuni isogenic mutants were obtained for use in future chicken challenge investigations and eukaryotic cell invasion assays. Information such as this will provide a basis for refining or adjusting intervention strategies to produce safer poultry food products, thereby reducing the risk of human exposure.
The accomplishment addresses Problem 1.1.3 (Ecology, Host Pathogen, and Chemical Contaminants Relationships) in the National Program 108 Food Safety 2006-2010 Action Plan for Pre-harvest Food Safety of Animals. The identification, at the molecular level, of factors involved in the colonization of poultry by zoonotic pathogens is critical for the development of targeted intervention strategies such that a safer food product can be delivered to consumers.
The accomplishment addresses Problems 1.1.1 (Methodology) and 1.1.3 (Ecology, Host Pathogen, and Chemical Contaminants Relationships) of the National Program 108 Food Safety 2006-2010 Action Plan for Pre-harvest Food Safety of Animals. The development of improved methods that accurately reproduce “in-plant” conditions provides for a greater understanding of the process of biofilm formation. Improved technology for artificial biofilm formation will facilitate the identification of environmental factors contributing to biofilm formation such that targeted intervention strategies can be developed.
Molecular characterization of zoonotic pathogens associated with poultry: In an effort to more accurately track the transmission of zoonotic pathogens through poultry production and processing environments, improved molecular subtyping technologies are needed. We continued the development of an expanded, more comprehensive repetitive extragenic palindromin-PCR (rep-PCR) fingerprint library (for Salmonella spp.) that will facilitate more accurate serotype designations for unknown Salmonella isolates to support epidemiologic analyses. The rep-PCR, utilizing a semi-automated procedure, was successfully evaluated as a method to determine Salmonella serotypes. This method may be utilized as an alternative to more time-consuming traditional serotyping methods for Salmonella spp.
The accomplishment addresses Problems 1.1.1 (Methodology) of the National Program 108 Food Safety 2006-2010 Action Plan for Pre-harvest Food Safety of Animals. The development of improved methods that accurately track pathogens from “farm to fork” will allow for improved understanding of the epidemiology of these pathogens in poultry. This improved understanding is necessary for the development and refinement of intervention strategies aimed at disruption of bacterial physiology will reduce these agents in poultry.
5.Significant Activities that Support Special Target Populations
|Number of Non-Peer Reviewed Presentations and Proceedings||4|
|Number of Newspaper Articles and Other Presentations for Non-Science Audiences||1|
Hiett, K.L., Stern, N.J., Cray, P.J., Cox Jr, N.A., Seal, B.S. 2007. Molecular phylogeny of the flaa short variable region (svr) among campylobacter spp isolates collected during an annual evaluatoin of poultry flocks in the southeastern united states. Foodborne Pathogens and Disease. 4(3):339-347.
Simmons, I.M., Hiett, K.L., Stern, N.J., Frank, J. 2008. Comparison of Poultry Exudate and Carcass Rinse Sampling Methods for the Recovery of Campylobacter spp. Subtypes Demonstrates Unique Subtypes Recovered from Exudate. Journal of Microbiological Methods. 74:89-93.
Hiett, K.L., Stintzi, A., Andacht, T.M., Kuntz, R.L., Seal, B.S. 2008. Genomic Difference between Campylobacter jejuni Isolates Identify Surface membrane and Flagellar Function Gene Products Potentially Important for Colonizing the Chicken Intestine.. Functional and Integrative Genomics. Epubed. 8(4):407-20.
Seal, B.S., Hiett, K.L., Kuntz, R.L., Woolsey, R., Schegg, K., Ard, M., Stintzi, A. Proteomic Analyses of a Robust versus a Poor Chicken Gastrointestinal Colonizing Isolate of Campylobacter jejuni. Journal of Proteome Research. 6:4582-4591.