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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

2009 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. Cell adhesion/invasion assays, using CaCo-2 cells and INT-407 cells with a library of 54 Campylobacter jejuni isolates, were completed to identify factors involved in colonization of broiler chickens by C. jejuni as well as factors contributing to human illness. Analyses demonstrated that C. jejuni isolates exhibit high genomic variability as well as significant variation in levels of adherence and invasiveness of human colonic cells and human intestinal cells. The results suggested that the genetic variation associated with C. jejuni might contribute to a varied level of eukaryotic cell adherence and invasion. Lipopolysaccharides (LPS) are an important determinant of host colonization. Bacterial LPS mutants for the acyl carrier (acpXL) and acyl transferase (lpxXL) genes were developed. Analysis of free-living, eukaryote associated, and post-infection bacterial LPS composition from wild type, an acpXL mutant, a lpxXL-like mutant, an acpXL,lpxXL double mutant, as well as plasmid complemented strains is currently being conducted. Analyses of the acpXL, lpxXL-like, and acpXL,lpxXL mutants of free-living bacteria revealed that the lipid-A long chain fatty acid found in wild type bacteria was absent from the mutant constructs. However, with respect to the eukaryote associated bacterial mutants, only the acpXL mutant was able to restore the long chain fatty acid. This suggests that the acyl transferase is required for the attachment of the long chain fatty acid whether carried by the AcpXL acyl carrier or an unidentified carrier. Microbial Ecology. An evaluation of levels of aerobic bacteria associated with non-washed and washed eggs was conducted. Analyses revealed that non-washed eggs produced by hens in cages (previously housed on shavings, slats, and cages) did not differ in aerobic plate counts (APC). However, when eggs were washed, a significant reduction (89%) in APC counts (0.2 log10 cfu/mL) was observed. Additionally, housing hens in cage units with manure removal and the absence of shavings/litter resulted in lower eggshell APC levels for both non-washed (by 2 log10 cfu/mL) and washed eggs (by 1 log10 cfu/mL) compared to eggs from hens housed in a room with cages, slats, and shavings. Corresponding reductions in human pathogens on eggshell surfaces would potentially improve food safety. Methods for the production of biofilms, comprised of Listeria monocytogenes, were developed and refined. The biofilms produced were used to screen various chemicals for their efficacy in biofilm disruption. The results of these experiments yielded an optimized formulation of a disinfectant, used for meat and poultry “ready to eat” (RTE) food processing equipment, that was more effective (defined as causing a total kill of >90%) relative to currently used disinfectants for reducing L. monocytogenes biofilm growth. Understanding the mechanism of action involved in biofilm disruption is critical for the future development of effective products against biofilms containing L. monocytogenes and other harmful pathogens.


4.Accomplishments
1. Colonization Factors. Cell adhesion/invasion assays, using a CaCo-2 cell line and a library of 54 Campylobacter jejuni isolates, were completed in an effort to identify factors involved in colonization of broiler chickens by C. jejuni as well as factors contributing to human illness. Completed analyses, including DNA:DNA microarray hybridization analyses and suppressive subtractive hybridization analyses) demonstrated that C. jejuni isolates exhibit high genomic variability as well as significant variation in the levels of adherence and invasiveness of transformed human colonic cells. The results suggested that the genetic variation associated with C. jejuni might contribute to varied level of eukaryotic cell adherence and invasion.

2. Colonization Factors. Phenotype microarray technology, a high throughput system used for the global analysis of cellular phenotypes, was employed for comparative analyses of C. jejuni and C. coli substrate utilization. Additionally, comparisons were conducted on a C. jejuni isolate propagated at 37°C and at 42°C. Preliminary results revealed several substrates that were differentially utilized between C. jejuni and C. coli. Additionally, several substrates were identified as being differentially utilized by C. jejuni at different growth temperatures.

3. Microbial Ecology. The evaluation of levels of aerobic bacteria associated with non-washed and washed eggs (obtained from caged and cage-free laying hens, housed on either shavings or wire slat floors) was conducted over an 8-month period. Analyses revealed that non-washed eggs produced by hens in cages (previously housed on shavings, slats, and cages) did not differ in aerobic plate counts (APC). Counts ranged from 0.67 to 0.84 log10 cfu/mL. However, when eggs were washed, a significant reduction (89%) in APC counts (0.2 log10 cfu/mL) was observed. Additionally, housing hens in cage units with manure removal and the absence of shavings/litter resulted in lower eggshell APC levels for both non-washed (by 2 log10 cfu/mL) and washed eggs (by 1 log10 cfu/mL) compared to eggs from hens housed in a room with cages, slats, and shavings. Corresponding reductions in human pathogens on eggshell surfaces would potentially improve food safety.

4. Microbial Ecology. Campylobacter spp. ecology in commercial Leghorn laying hens was evaluated by determining.
1)Campylobacter spp. presence in the reproductive tract, lymphoid organs, liver/gallbladder, and ceca of hens,.
2)the species of Campylobacter present, and.
3)the antibiotic resistance patterns to 9 antimicrobials. The recovery rate of Campylobacter spp. was 13%, 67%, 53%, 3%, 13%, and 57% from the ovarian follicles, lower reproductive tract, upper reproductive tract, spleen, liver/gallbladder and ceca, respectively; a repetition of the investigation resulted in similar rates of recovery. Overall, 50% of the isolates were speciated as C. jejuni, 49% C. coli, and 1% C. lari. Additionally in the first investigation, all recovered isolates were pan susceptible to the antimicrobials while in the second investigation 37% of the isolates were resistant to tetracycline.

5. Microbial Ecology. A C. coli isolate resistant to gentamicin at >100 µg/ml was identified. In an effort to facilitate chick challenge investigations, the identified C. coli isolate was inoculated into chicks. Inoculated broilers were removed at several intervals and a variety of tissue types were sampled for the presence of the marker strain. Results demonstrated that the marker C. coli isolate colonized chicks, disseminated to body tissues, colonized penmates and persisted throughout the 6-week broiler production period.

6. Microbial Ecology. A survey to determine the presence of Campylobacter spp. in free-ranging birds was conducted in the Southeastern United States. Thirteen samples from birds with omnivorous diets, were positive for Campylobacter spp.-like organisms using a filtration/BAB methodology. Campylobacter spp. identified phenotypically included only C. jejuni and C. coli while several recovered colonies had rRNA DNA sequences consistent with Helicobacter spp. Three of the recovered isolates demonstrated resistance to at least 1 of the 9 antimicrobials tested.


Review Publications
Callicott, K., Haroardottir, H., Georgsson, F., Reiersen, J., Frioriksdottir, V., Gunnarsson, E., Michel, P., Bisaillon, J., Kristinsson, K.G., Briem, H., Hiett, K.L., Needleman, D.S., Stern, N.J. 2008. Broiler Contamination and Human campylobacteriosis in Iceland. Applied and Environmental Microbiology. 74(21):6483-6494.

Wise, M., Siragusa, G.R., Plumblee, J., Healy, M., Cray, P.J., Seal, B.S. 2009. Predicting Salmonella enterica serotypes by repetitive sequence-based PCR. Journal of Microbiological Methods. 76(1):18-24.

Cox Jr, N.A., Richardson, L.J., Berrang, M.E., Cray, P.J., Buhr, R.J. 2009. Campylobacter coli naturally resistant to elevated levels of gentamicin as a marker strain in poultry research. Journal of Food Protection. 72(6):1288-1292.

Hannah, J.F., Fletcher, D.L., Cox Jr, N.A., Smith, D.P., Cason Jr, J.A., Northcutt, J.K., Richardson, L.J., Buhr, R.J. 2009. Impact of Added Sand on the Recovery of Salmonella, Campylobacter, Escherichia coli, and Coliforms from Pre-Chill and Post-Chill Commercial Broiler Carcass Halves. Journal of Applied Poultry Research. 18:(2)252-258.

Northcutt, J.K., Mcneal, W.D., Ingram, K.D., Buhr, R.J., Fletcher, D.L. 2008. Bacteria recovery from genetically featherless broiler carcasses after forced cloacal fecal expulsion. Poultry Science. 87:(11) 2377-2381.

Roche, A.J., Cox Jr, N.A., Richardson, L.J., Buhr, R.J., Cason Jr, J.A., Fairchild, B.D., Hinkle, N.C. 2009. Contaminated Larval and Adult Lesser Mealworms, Alphitobius diaperinus (Coleoptera: Tenebrionidae)can Transmit Salmonella Typhimurium in a Broiler Flock. Poultry Science. 88:44-48.

Arnold, J.W. 2008. Colorimetric assay for bioWlms in wet processing conditions. Journal of Industrial Microbiology and Biotechnology. 35(11):1475-1480.

Arnold, J.W., Yates, I.E. 2009. Interventions for control of Salmonella: clearance of microbial growth from rubber picker fingers. Poultry Science. 88(6):1292-1298.

Edwards, J.V., Caston-Pierre, S., Howley, P.S., Condon, B.D., Arnold, J.W. 2008. A bio-sensor for human neutrophil elastase employs peptide-p-nitroanilide cellulose conjugates. Sensor Letters. 6(4):518-523.

Siragusa, G.R., Haas, G.J., Matthews, P.D., Smith, R.J., Buhr, R.J., Dale, N.M., Wise, M.G. 2008. Antimicrobial Activity of Lupulone against Clostridium perfringens in the Chicken Intestinal Tract Jejenum and Caecum. Antimicrobial Chemotherapy. 61(4):853-858.

Hong, Y.H., Lillehoj, H.S., Siragusa, G.R., Bannerman, D.D., Lillehoj, E.P. 2008. Antimicrobial activity of chicken NK-lysin against Eimeria sporozoites. Avian Diseases. 52:302-305.

Fu, J., Park, B., Siragusa, G.R., Jones, L., Tripp, R.A., Zhao, Y., Cho, Y. 2008. Au/Si Hetero-Nanorod-based Biosensor for Salmonella Detection. Nanotechnoloyg 19: 155502.

Park, B., Fu, J., Zhao, Y., Siragusa, G.R., Cho, Y., Lawrence, K.C., Windham, W.R. 2007. Bio-Functional Au/Si nanorods for Pathogen Detection. Proceedings of SPIE 6769-26.

Barrios, P.R., Reiersen, J., Lowman, R., Bisaillon, J.R., Michel, P., Fridriksdottir, V., Gunnarsson, E., Stern, N.J., Berke, O., Mcewen, S., Martin, W. 2006. Risk factors for campylobacter spp. colonization in broiler flocks in iceland. Preventive Veterinary Medicine. 74(4):264-278.

Li, X., Swaggerty, C.L., Kogut, M.H., Chiang, H., Wang, Y., Genovese, K.J., He, H., Stern, N.J., Pevzner, I.Y., Zhou, H. 2008. The paternal effect of Campylobacter jejuni colonization in ceca in broilers. Poultry Science. 87:1742-1747.

Last Modified: 9/10/2014
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