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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

2010 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
Colonization Factors: Eukaryotic cell (human colonic and human intestinal) adhesion/invasion assays were performed on fifty-one C. jejuni isolates. A range of adhesion/invasion abilities was exhibited. Isolates within the human isolate source group demonstrated a significantly higher mean % adherence to human colonic cells than did isolates within the broiler source group. In an effort to identify genes involved in C. jejuni virulence, comparative genomic analyses were performed on the most invasive isolate, the least invasive isolate, and 2 isolates in the middle of the observed range. A high degree of genetic variability, generally located within 7 previously defined plasticity regions, was observed. The greatest variability occurred in the functional categories of cell envelope/surface structures, small molecule metabolism, and hypothetical/genes of unknown function. The research led to the identification of two genes that may be indicative of the ability to adhere to and subsequently invade human cell lines. These genes may ultimately serve as biomarkers to allow for the identification of C. jejuni isolates more likely to be involved with human illness. Additionally, this research led to the identification of a gene that is currently being developed as fusion vaccine against C. jejuni.

Campylobacter spp. exhibit unique nutritional requirements. C. jejuni is most often associated with poultry, while C. coli are more frequently associated with swine. Temperature has been suggested to trigger potential colonization or virulence factors in C. jejuni and recent studies have demonstrated temperature-dependent genes are important to colonization. It is possible that temperature-dependent colonization factors are in part responsible for the species-specific colonization characteristics of C. coli also. We determined utilization of 190 different sole carbon substrates by C. coli at 37C and 42C using phenotype microarray (PM) technology. Temperature was determined to affect amino acid utilization. Further investigation is needed to determine the basis for the temperature-dependent utilization of substrates by Campylobacter spp. and their possible role in species-specific colonization. Microbial Ecology: Campylobacter spp. are frequently present in the intestinal tract and internal tissues of broiler breeder and broiler chickens. Campylobacter spp. ecology in commercial Leghorn laying hens had not been extensively studied. Current research study revealed a high prevalence of C. jejuni and C. coli in the 5 flocks evaluated from 2 commercial laying operations. Presence in organs and tissues within Leghorn hens was similar to that found in broilers and broilers breeders, even after the Leghorn hens spent several months in a caged environment. Tetracycline-resistant Campylobacter was most often observed. Knowledge of the ecology of these organisms in laying operations is critical for evaluation of transmission to neighboring poultry flocks.


4.Accomplishments
1. Identification of Unique Salmonella Subtypes Recovered Using Novel Cultivation Media: Using cultivation methods, not commonly employed by regulatory agencies for the isolation of Salmonella from poultry, ARS scientists in Athens, GA recovered 176 Salmonella isolates from 49 post-pick carcass rinse samples. Subtype analysis (XbaI PFGE) and subsequent comparison of the subtypes to those in the USDA-Vet Net data base (4,620 patterns from 17,597 isolates) revealed 10 new Salmonella patterns with 6 of these patterns originating from the 57 S. Kentucky isolates. Additionally, analysis of the 28 S. Kiambu isolates recovered, resulted in a unique pattern showing a slight band shift difference from all other S. Kiambu found previously on chicken; this unique pattern was only observed twice before in diagnostic cattle samples. The identification of new patterns suggests that further work needs to be conducted on cultural influences that select certain Salmonella serotypes along with diversity within serotypes.

2. Identification of Genes Involved in C. jejuni Colonization of Poultry and Genes that May Serve as Biomarkers for Human Disease: Campylobacter spp., commonly associated with poultry, are considered the leading cause of human bacterial food-borne illness in the United States. Comparative genomic analyses of 4 C. jejuni isolates (recovered from different hosts and demonstrating different levels of eukaryotic adhesion/invasion values) identified several genes potentially involved in C. jejuni colonization of broiler chickens. One of the identified genes is currently being developed as a fusion vaccine against C. jejuni during poultry production. Additionally, several genes that may be indicative of the ability to adhere to and subsequently invade human cells were also identified. These latter genes may ultimately serve as biomarkers that allow for the identification of C. jejuni isolates more likely to be involved with human illness.

3. Temperature Affects Sole Carbon Utilization Patterns of Campylobacter coli 49941: Campylobacter spp. are small bacteria incapable of metabolizing glucose. C. jejuni is most often recovered from poultry, while C. coli are more frequently recovered from swine. Temperature has been suggested to trigger potential colonization or virulence factors in C. jejuni; recent studies support this suggestion as it was demonstrated that temperature-dependent genes are important to colonization. It is possible that temperature-dependent colonization factors are in part responsible for the species-specific colonization characteristics of C. coli as well. ARS scientists in Athens, GA determined the utilization of 190 different sole carbon substrates by C. coli ATCC 49941 at 37°C and 42°C using phenotype microarray (PM) technology. Temperature was shown to significantly affect utilization of specific amino acids. Knowledge of temperature-dependent substrate utilization by different Campylobacter spp. will provide information regarding species-specific colonization. This information could lead to improvements in culture media for detection and isolation of the pathogen as well as to future intervention methods that ultimately reduce human exposure.

4. Recovery of Surface and Deep Eggshell Aerobic Bacteria from Commercial Broiler Hatching Eggs Using Rinse and Crush-and-Rub Methods after Hatching Egg Sanitization: Hatching egg disinfection prior to setting appears to reduce eggshell bacteria levels to different degrees dependent upon the sampling method used (Rinse “R” and Crush-and-Rub “CR”). Hatching eggs were placed into 5 treatments (no treatment = Control, Water, Hydrogen Peroxide (H2O2), Phenol, and a combination chemical (4 quaternary ammoniums + 1 biguanide “Q4B”). Aerobic bacteria recovery levels from the Control (4.0 R and 2.5 CR log10cfu/mL of eggshell rinsate) and Water (3.7 R and 2.3 CR) treatments were not significantly different. Bacteria recovery levels from the Phenol (3.2 R and 1.6 CR) and H2O2 (3.1 R and 1.9 CR) treatments were similar and both were significantly lower than the Control and Water treatments. Bacteria recovery levels from the Q4B treatment (2.4 R and 0.9 CR) were significantly lower than all other treatments. The overall correlation between the rinse and crush-and-rub aerobic counts was high at r = 0.71. Individual treatment group correlations were: Water 0.72, H2O2 0.67, Phenol 0.73, and Q4B 0.38. The similar correlations for Water, H2O2, and Phenol indicate the main impact was from wetting the eggshell. The lower correlation for Q4B indicates larger differences in bacteria recover levels from rinse and crush-and-rub methods indicating bactericidal ability. These data suggest that the compound chemical (Q4B) is a very effective sanitizer on broiler hatching eggs and trials evaluating impact on hatchability should be conducted.

5. Genotype Analysis of Campylobacter spp. Recovered from Human Disease and Animals Other than Broiler Chickens in Iceland (2001-2004): Several investigations on the transmission of Campylobacter spp. through the environment, agricultural animals, and eventually to humans have been conducted. However, the most critical sources involved in transmission continue to remain unclear. As a component of an extensive epidemiologic investigation conducted on Campylobacter spp. transmission in Iceland, ARS scientists in Athens, GA completed molecular subtype studies to establish the role of non-broiler chicken sources (such as cattle, ducks, sheep, and swine) in the transmission of Campylobacter spp. to poultry flocks as well as to humans. Campylobacter spp. isolates recovered from agricultural animals, other than broiler chickens, were determined to possess Campylobacter spp. isolates with identical subtypes to those isolates involved in human infection. Additionally, temporal relationships were apparent. Identification of specific subtypes involved in human clinical infections will allow for further analyses such that biomarkers associated with human infection may be identified.


Review Publications
Cox Jr, N.A., Richardson, L.J., Musgrove, M.T. 2010. Campylobacter jejuni, other campylobacters. Chapter 3 In Pathogens and Toxins in Foods: Challenges and Interventions. P.20-30.

Cox Jr, N.A., Richardson, L.J., Buhr, R.J., Cray, P.J. 2009. Campylobacter spcies occurrence within internal organs and tissues of commercial caged Leghorn laying hens. Poultry Science. 88(1):2449-2456.

Bailey, J.S., Richardson, L.J., Cox Jr, N.A., Cosby, D.E. 2010. Salmonella. Pathogens and Toxins in Foods: Challenges and Interventions. Chapter 7:108-118.

Cox Jr, N.A., Richardson, L.J., Cason Jr, J.A., Buhr, R.J., Vizzier-Thaxton, Y., Smith, D.P., Cray, P.J., Romanenghi, C.P., Pereira, L.B., Doyle, M.P. 2010. Comparison of neck skin excision and whole carcass rinse sampling methods for determining Salmonella prevalence and E. coli counts on broiler carcasses before and after immersion chilling. Journal of Food Protection. 73(5):976-980.

Hiett, K.L., Seal, B.S. 2009. Use of Repetitive Element Palindromic-PCR (rep-PCR) for the Epidemiologic Discrimination of Food-Borne Pathogens. Methods in Molecular Biology. Volume number 551 pages 49-58.

Last Modified: 9/10/2014
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