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United States Department of Agriculture

Agricultural Research Service

2009 Annual Report

1a.Objectives (from AD-416)
The purpose of the Poultry Microbiological Safety Research Unit is to further reduce or eliminate bacterial pathogen contamination in poultry operations and the following objectives are to:.
1)Assess the effectiveness and further development of bacteriocins (anti-bacterial peptides) and bacteriophage by in vitro bacterial growth inhibition in culture and in vivo experimentation via challenge in chickens..
2)Reduce bacterial populations in chicken litter by monitoring poultry houses for bacterial pathogens carried by chickens during use of intervention technologies. Decontamination techniques, such as in-house foam applied disinfectants will be examined for reduction of Campylobacter spp., Salmonella spp., Clostridium perfringens and Listeria monocytogenes..
3)Improve cultural methods for Campylobacter spp. in poultry in further support of FSIS needs, including the improvement of recovery. Use microarray expression analysis under various cultural conditions to identify nutrients necessary for optimal growth, colonization and culture of Campylobacter spp.

1b.Approach (from AD-416)
New antimicrobial agents will be developed that are usable by the poultry farmer on a large scale to reduce Campylobacter spp. and salmonellae by applying in feed at the appropriate times before slaughter. Also, new means for the drug-free production sector to control clostridial disease without relying on antibiotics are the major expected approaches of Objective 1. Providing a science based source of data to better dispose of spent poultry litter without major energy or financial inputs is the approach for Objective 2. A more reliable and quantitative method to routinely culture Campylobacter spp. from food and environmental samples is the approach of Objective 3.

3.Progress Report
Bacteriocin isolation and production. Control of both Campylobacter and Salmonella has been demonstrated using bacteriocins in live poultry. Successful commercial application will require production of large quantities of bacteriocins. We determined that a combination of producer and inducer bacterial strains and signal peptides could markedly increase production. A related manuscript was submitted this year. Antagonists against Clostridium perfringens. Investigations continued by identifying bacteriophage enzymes that lyse Clostridium perfringens. Additional lytic genes were identified from presumed prophage and host encoded bacterial DNA. One lysin gene was isolated from Clostridium perfringens and a second gene was cloned from Listeria monocytogenes genomic DNA. Both genes were expressed as recombinant proteins and demonstrated to lyse the parent bacterium. Assessment of the chemical and microbial impact of poultry litter. The horizontal transmission of Salmonella and Campylobacter was evaluated for caged and cage-free housed hens. Challenged hens were comingled with non-challenged hens in conventional colony cages, on wire slats, or on shavings flooring systems. The horizontal spread of Salmonella was similar among non-challenged hens across housing systems (31-38%). However, Campylobacter was recovered from 33% of the non-challenged hens on litter compared to 0% and 3% of the hens housed in cage or wire slat systems. Examination of chemical disinfection techniques for reduction of pathogens. Evaluation of the superficial and deep eggshell bacteriology of hatching eggs sanitized in a commercial hatchery after either spray or foam sanitization was conducted. An eggshell surface rinsate and a crush-and-rub rinsate were evaluated for aerobic bacteria, Escherichia coli, and coliforms. Results indicate that sanitization of hatching eggs by spray or foam significantly reduces the level of bacteria on the eggshell surface recovered in a rinse, but spray or foam sanitization did not alter the level of bacteria within the eggshell/membranes recovered in a crush-and-rub rinse method. Improved cultural methods for pathogen recovery. The U.S. Food Safety Inspection Service (FSIS) utilizes a rinse technique for detection of Salmonella on broiler chicken carcasses, while the European Union (EU) uses composited neck skin samples. Salmonella detection on commercial broiler carcasses was compared using both methods. There was no significant difference in Salmonella incidence observed between the FSIS rinse method (32/60) and the EU method (30/60). However, a combination of both procedures did increase the determination of Salmonella prevalence (48/60) on carcasses. Phenotype microarray methodology has been optimized and experiments on carbon utilization have been conducted with different species of Campylobacter at different incubation temperatures. Results will determine unique cultural requirements necessary for improved detection, growth and recovery of the pathogens.

1. Antimicrobial activities of bacteriocins against hospital acquired infections. Bacteriocins are non-toxic proteins produced by bacteria that kill other bacteria. We used bacteriocins to study antibacterial activities against antibiotic resistant Staphylococcus aureus, and other prominent hospital acquired bacterial pathogens (Acinetobacter baumannii; Citrobacter freundii; Escherichia coli; Klebsiella pneumoniae; Proteus spp; and Pseudomonas aeruginosa) collected from Russian hospitals during 2003-2007. Antimicrobial resistance was determined and measured by minimal inhibitory concentrations (MICs). The MIC values of the same isolates ranged from < 0.025 to 1.56 mg/ml for bacteriocin B 602 and 0.05 to 6.25 mg/ml for bacteriocin E 50-52. For the 64 diverse clinical isolates tested, therapeutic antibiotic resistance was high while the susceptibility to the bacteriocins tested was remarkably good. The potentials for application of bacteriocins in clinical settings as therapeutic agents appear quite promising.

2. Inducer bacteria, signal proteins and low nutrient media stimulate bacteriocin production. We previously reported on three bacteriocins produced by Lactobacillus salivarius B-30514 (OR-7), Enterococcus faecium B-30746 (E 50-52) and Enterococcus spp. B-30745 (E 760). In an infected adult chicken model, milligram quantities of bacteriocin reduced Campylobacter jejuni gastrointestinal colonization by a million-fold. To commercially apply such bacteriocins, adequate quantities must be produced and harvested. We determined that a combination of signal peptides in the presence of both producer and selected inducer (Lactobacillus acidophilus and Lactobacillus crispatus) bacteria grown in a dilute fermentation medium enabled marked increase in synthesis of bacteriocin. We used a starvation medium of 10% brucella broth to enhance further enhance production. Using the combination of these three parameters enabled us to significantly increase the yield of these bacteriocins so they can now be efficiently produced in commercial quantities.

3. Clearance of Salmonella from rubber picker fingers used to defeather poultry. A study was conducted to determine if a surface material with antimicrobial properties combined with an effective disinfectant could achieve total clearance of bacterial contamination from rubber picker fingers that are used to defeather poultry. Five disinfectants were tested separately for effectiveness against Salmonella growing on rubber picker fingers. The results indicated that Salmonella could be cleared completely by a nontoxic compound, which alleviates concerns for the health of employees in processing facilities and reduces the negative impact of agriculture on the environment.

6.Technology Transfer


Review Publications
Svetoch, E.A., Levchuk, V.P., Pokhilenko, V.D., Eruslanov, B.V., Mitsevich, E.V., Mitsevich, I.P., Perelygin, V.V., Stepanshin, Y.G., Stern, N.J. 2008. Killing of Methicillin Resistant Staphylococcus aureus (MRSA) and other Pathogens by Bacteriocins OR-7 and E 50-52. Journal of Clinical Microbiology. 46(11): 3863-3865.

Yoon, S.C., Lawrence, K.C., Siragusa, G.R., Line, J.E., Park, B., Feldner, P.W. 2009. Hyperspectral Reflectance Imaging for Detecting a Foodborne Pathogen: Campylobacter. Transactions of the ASABE. 52(2): 651-662

Richardson, L.J., Cox Jr, N.A., Bailey, J.S., Berrang, M.E., Cox, J.M., Buhr, R.J., Cray, P.J., Harrison, M.A. 2009. Evaluation of TECRA® broth, Bolton broth and direct plating for recovery of Campylobacter spp, from broiler carcass rinsates from several commercial processing plants. Journal of Food Protection. 72(5):972-977.

Musgrove, M.T., Northcutt, J.K., Jones, D.R., Cox Jr, N.A., Harrison, M.A. 2008. Enterobacteriaceae and Related Organisms Isolated from Shell Eggs Collected During Commercial Processing. Poultry Science. 87:1211-1218.

Bailey, J.S., Cray, P.J., Richardson, L.J., Cox Jr, N.A., Cox, J.M. 2008. Detection of Campylobacter from broiler carcass rinse samples utilizing the TECRA Visual Immunoassay (VIA). Journal of Rapid Methods and Automation in Microbiology.16(4):374-380.

Line, J.E., Hiett, K.L. 2008. Comparison of Challenge Models for Determining the Colonization Dose of Campylobacter jejuni in Broiler Chicks. Poultry Science. 87:1700-1706.

Briem, H., Fridriksdottir, V., Georgsson, V., Gunnarsson, E., Hardardottir, H., Kristinsson, K., Sigmundsdottir, G., Reiersen, J., Berndtson, E., Bisaillon, J.R., Fazil, A., Michel, P., Paoli, G., Lowman, R., Callicott, K.A., Hiett, K.L., Stern, N.J. 2008. Temperature-related risk factors associated with the colonization of broiler-chicken flocks with Campylobacter spp. in Iceland, 2001-2004. Preventive Veterinary Medicine. 86(1-2):14-29.

Stern, N.J., Line, J.E. 2009. Enmeration of Campylobacter spp., Escherichia coli, and Salmonella in broiler carcass rinses before and after simulated transport in artificial ice for 24 hours. Journal of Food Protection. 72(5):1099-1101.

Wakamatsu, N., King, D.J., Seal, B.S., Peeters, B.H., Brown, C. 2006. The effect on pathogenesis of Newcastle disease virus LaSota strain from a mutation of fusion cleavage site to a virulent sequence. Avian Diseases. 50:483-488.

Wakamatsu, N., King, D.J., Seal, B.S., Samal, S.K., Brown, C.C. 2006. The pathogenesis of Newcastle disease: A comparison of selected Newcastle disease virus wild-type strains and their infectious clones. Virology. 353:333-343.

Wakamatsu, N., King, D.J., Seal, B.S., Brown, C.C. 2007. Detection of Newcastle disease virus RNA by reverse transcription polymerase chain reaction using formalin-fixed, paraffin-embedded tissue and comparison with immunohistochemistry and in situ hybridization. Journal of Veterinary Diagnostic Investigation. 19:396-400.

Estevez, C., King, D.J., Seal, B.S., Yu, Q. 2007. Evaluation of Newcastle disease virus chimeras expressing the hemagglutinin-neuraminidase protein of velogenic strains in the context of a mesogenic recombinant virus backbone. Virus Research. 129:182-190.

Stern, N.J., Svetoch, E.A., Eruslanov, B.V., Perelygin, V.V., Mitsevich, E.V., Mitsevich, I.P., Pokhilenko, V.D., Levchuk, V.P., Svetoch, O.E., Seal, B.S. 2006. Isolation of a lactobacillus salivarius: its bacteriocin is inhibitory to campylobacter jejuni in chickens. Antimicrobial Agents and Chemotherapy. Vol 50:3111-3116.

Stern, N.J. 2008. Salmonella species and Campylobacter jejuni Cecal Colonization Model in Broilers. Poultry Science. 87(11):2399-2403.

Snelling, W.J., Stern, N.J., Lowery, C.J., Moore, J.3., Gibbons, E., Ciara, B., Dooley, J.S. 2007. Colonization of broilers by Campylobacter jejuni internalized within Acanthamoeba castellanii. Archives Of Microbiology. DOI 10.107/s00203-007-0303-0.

Last Modified: 8/27/2015
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