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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR CHARACTERIZATION AND GASTROINTESTINAL TRACT ECOLOGY OF COMMENSAL HUMAN FOOD-BORNE BACTERIAL PATHOGENS IN THE CHICKEN

Location: Poultry Microbiological Safety Research

2007 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.


3.Progress Report
The methods of suppressive subtractive hybridization and microarray hybridization along with proteomics analyses were used to determine gene content of two C. jejuni isolates with different abilities for chicken colonization. This revealed that the specific expression of an outer membrane-fibronectin binding protein, serine protease and a putative aminopeptidase in the soluble portion of the robust colonizer A74C. Several proteins including a cysteine synthase and aconitate hydratase were detected specifically in the poor colonizer C. jejuni NCTC 11168-PMSRU isolate. Interestingly, different gene products potentially involved in robust colonization of chickens by Campylobacter spp. appear to conform to recently identified expression patterns in biofilm or agar adapted isolates. Among commercial parent breeder flocks imported into Iceland poultry production that were hatched from eggs coming from Campylobacter-positive grandparent flocks no evidence for vertical transmission of Campylobacter was found in the production flocks. A crystal violet assay for measurement of bacterial biofilms on stainless steel was developed that could be applicable to a wide variety bacteria and substrata.


4.Accomplishments
1. Identification of differential gene content and protein expression of C. jejuni isolates demonstrated to differentially colonize chickens The identification of genes and gene products involved in Campylobacter spp. colonization of chickens is necessary for the development of intervention strategies aimed at the reduction or elimination of Campylobacter spp. in poultry. Therefore, suppressive subtractive hybridization and microarray hybridization were used to determine gene content of two C. jejuni isolates with different abilities for chicken colonization. Additionally, differencesin protein expression was investigated using 2-dimensional gel electrophoresis. It was determined that a variety of genes and proteins expressed by the good chicken colonizing C. jejuni isolate were not present in the poor colonizing isolate. A majority of theses genes were located in areas of the C. jejuni genome previously determined to be hyper-variable. Consequently, if interventions can be developed that disrupt specific biological functions of C. jejuni, such as attachment of the bacteria to chicken gastrointestinal cells, these bacteria could be reduced in chickens prior to processing for human consumption. The accomplishment addresses Problem 1.1.3 of the Ecology, Host Pathogen and Chemical Residue Relationships in the National Program 108 Food Safety 2006-2010 Action Plan for Preharvest Food Safety of Animals. It is not sufficient to know that food animals may be carrying one or more specific epizootic pathogens; we need to know their relationship to and the attributes of the microbial communities in which pathogens live, particularly the gut of food producing animals. This is 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)

2. Characterization of Campylobacter spp. ecology and molecular ecology of the chicken gastrointestinal tract. It was demonstrated that there was a lack of evidence for vertical transmission of Campylobacter spp. in poultry during processing of chickens. Among approximately 60,000 progeny parent breeders that were hatched from eggs coming from Campylobacter-positive grandparent flocks no evidence for vertical transmission of Campylobacter was found. The effect of drug-free poultry production on the intestinal microflora of broiler chickens, the bacterial community of this environment was quantitatively profiled in both conventionally reared birds and birds reared without antibiotic growth promotants (AGPs). Quantitative, real-time PCR indicated that the effects of drug-free rearing on the chicken GI tract microbial community are most pronounced in the ileal region, but AGPs may be important in controlling Campylobacter colonization of the caecum. The accomplishment addresses Problem 1.1.3 of the Ecology, Host Pathogen and Chemical Residue Relationships in the National Program 108 Food Safety 2006-2010 Action Plan for Preharvest Food Safety of Animals. It is not sufficient to know that food animals may be carrying one or more specific epizootic pathogens; we need to know their relationship to and the attributes of the microbial communities in which pathogens live, particularly the gut of food producing animals. This is 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)

3. Development of methods to quantitatively assess L. monocytogenes under conditions that mimic poultry environments. The bacterial pathogen, Listeria monocytogenes, causes a high death rate among its victims and many food product recalls. In an effort to develop methods to quantitatively assess L. monocytogenes under conditions that mimic poultry environments. Samples were measured separately for stainless steel coupons by aerobic plate counts, a modified crystal violet assay, and spectrophotometry. L. monocytogenes formed biofilms on all substrata tested. This study will provide data for risk assessment of L. monocyotogenes. This is the first use of the crystal violet assay for measurement of bacterial biofilms on stainless steel. The methods tested are applicable to other bacteria and substrata. The accomplishment addresses Problem 1.1.1 Methodology in the National Program 108 Food Safety 2006-2010 Action Plan for Preharvest Food Safety of Animals. Rapid, accurate, and sensitive methodology is needed for identification and quantitation of epizootic pathogens and chemicals that may be transmitted to humans, as well as for other bacteria that may affect their occurrence in the animal and their environment. This is 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 1.1.1 (Methodology).


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of active CRADAs and MTAs3
Number of non-peer reviewed presentations and proceedings14

Review Publications
Manfreda, G., Decesare, A., Bondioli, V., Stern, N.J., Franchini, A. 2006. Enumeration and identity of campylobacter spp. in italian broilers. Poultry Science. (85):556-562.

Callicott, K., Fridriksdottir, V., Reiersen, J., Lowman, R., Bisaillon, J., Gunnarsson, E., Berndtson, E., Hiett, K.L., Needleman, D.S., Stern, N.J. 2006. Lack of evidence for verticle transmission of campylobacter spp. in chickens. Applied and Environmental Microbiology. 72(9):5794-5798.

Hiett, K.L., Siragusa, G.R., Seal, B.S. 2006. Discrimination of campylobacter spp. by use of gel-based rep-pcr as compared with flaa gene short variable region dna sequence analysis. Journal of Applied Microbiology. 3:50-54.

Siragusa, G.R., Wise, M. 2007. Quantitative analysis of the gastrointestinal bacterial community of broiler chickens reared with and without antimicrobial growth promotants. Journal of Applied Microbiology. 102(4):1138-49.

Stern, N.J., Georgsson, F., Lowman, R., Bisaillon, J.R., Reiersen, J., Callicott, K., Geirsdottir, M., Hrolfsdottir, R., Hiett, K.L. Frequency and enumeration of campylobacter spp. from proceeded broiler carcasses using weep and rinse samples. Poultry Science. 86:394-399.

Sanders, S.Q., Boothe, D.D., Frank, J., Arnold, J.W. Culture and detection of campylobacter jejuni within mixed populations of biofilms on stainless steel. Journal of Food Protection. Vol. 70:6, p1379-1385.

Last Modified: 4/21/2014
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