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United States Department of Agriculture

Agricultural Research Service

Research Project: INTERVENTIONS AND METHODOLOGIES TO REDUCE HUMAN FOOD-BORNE BACTERIAL PATHOGENS IN CHICKENS

Location: Poultry Microbiological Safety Research

2008 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
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).

Bacteriocin isolation and production. Additional Campylobacter antagonistic bacteria have been identified and their corresponding bacteriocins isolated and characterized. Control of both Campylobacter and Salmonella has been demonstrated using the newly isolated bacteriocins in live poultry. We have reduced the level of Campylobacter in the ceca of chickens by at least one-million fold via oral treatment of broilers with specific bacteriocins. By reducing the levels of pathogens in the broiler intestinal tract, corresponding cross contamination of poultry meat will be proportionately diminished and consumer exposure to these pathogens will be decreased. Two manuscripts and a patent have been published this year.

Antagonists against Clostridium perfringens. We continued carrying out the research, despite losing a key Senior Scientist position this year. Screening for bacteriophages lytic for Clostridium perfringens was completed utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. From the collection a highly lytic virus was isolated. The double-stranded deoxyribonucleic acid (DNA) genome of the virus was sequenced to completion and several bacteriophage genes were identified. DNA sequencing of a bacteriophage genome completed in collaboration with Russian investigators resulted in identification of a unique amidase as well as a phage encoded protein that potentially contains lysozyme and endopeptidase activities.

Assessment of the chemical and microbial impact of poultry litter. A manuscript was published describing the effect of rainfall and tillage on transport of fecal bacteria from broiler litter applications.

Improved cultural methods for Campylobacter recovery. A manuscript was published describing the addition of sulfamethoxazole to selective media to aid in the recovery of Campylobacter bacteria from broiler carcases.

Phenotype microarray methodology has been optimized and experiments have been conducted this year with different species of Campylobacter to help determine unique cultural requirements necessary for detection, growth and recovery of the pathogens.

Two Campylobacter enrichment broths and twelve different enrichment procedures (using the two broths) were evaluated for their sensitivity to recover Campylobacter from rehang and post-chill commercial carcass rinses. Results demonstrated the lack of cultural sensitivity by certain methods for recovery of injured, stressed, or low numbers of Campylobacter cells. Increased sensitivity for recovery of these types of cells was observed when using a combination of enrichment broth methods.


4.Accomplishments
1. Diverse antimicrobial killing by Enterococcus faecium E 50-52 bacteriocin. Campylobacter jejuni is a Gram-negative human food-borne pathogen of primary importance. Poultry are frequently contaminated with C. jejuni during production with the majority of commercial U.S. flocks positive for the organism by the time the birds reach market age at about 6 weeks. There is a need for effective intervention that may be practically applied in the poultry industry to reduce colonization of poultry with C. jejuni and subsequently reduce consumer exposure to this pathogen. An effective bacteriocin (E 50-52) was identified and characterized, and demonstrated MIC’s against C. jejuni, Yersinia spp., Salmonella spp., E. coli O157:H7, Shigella dysenteriae, Morganella morganii, Staphylococcus spp. and Listeria spp. ranging from 0.025 to 32 µg/ml. In therapeutic broiler trials oral treatment with E 50-52 reduced both Campylobacter jejuni and Salmonella enteritidis by >100,000-fold in the ceca and systemic S. enteritidis was reduced in the liver and spleen. Treatments with viable probiotic bacterial cultures were ineffective in reducing C. jejuni in chickens while bacteriocin treatment from these corresponding bacteria substantially reduced C. jejuni colonization in the live birds. Bacteriocin E 50-52 could be developed as a practical on-farm intervention for reducing consumer exposure to pathogenic campylobacter and such information will be useful to researchers in government, academia and the poultry industry. 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).

2. Bactericidal effect of several chemicals on hatching eggs inoculated with Salmonella serovar Typhimurium. Bactericidal effects of polymers in combination with biocides were studied as a means to sanitize broiler hatching eggs. Breeder flocks and commercial hatcheries represent an early contamination point for Salmonella entry into commercial integrated poultry operations. Several of the commercial antimicrobial chemicals were found to significantly reduce or eliminate Salmonella on fertile hatching eggs in laboratory experiments. The chemicals that totally eliminated Salmonella on the fertile hatching egg were used and effect on hatchability tested. No significant differences were observed in hatchability from eggs sprayed with or without the chemical polymers in combination with biocides, indicating no adverse effects on livability of developing broiler chicks. The successful chemicals are now being evaluated in a commercial hatchery. Using effective antimicrobial treatments for hatching eggs is a critical part of reducing on farm incidence of Salmonella. 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).


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of Invention Disclosures Submitted1
Number of Non-Peer Reviewed Presentations and Proceedings6

Review Publications
Svetoch, E.A., Eruslanov, B.V., Perelygin, V.V., Vitsevich, E.V., Mitsevich, I.P., Borzenkov, V.N., Levchuk, V.P., Svetoch, O.E., Kovalev, Y.N., Stephanshin, Y.G., Siragusa, G.R., Seal, B.S., Stern, N.J. 2008. Diverse antimicrobial activity from Enterococcus faecium NRRL B-30746 bacteriocin. Journal of Agric Food Chem. 56(6), 1942-1948.

Jenkins, M., Truman, C.C., Siragusa, G.R., Line, J.E., Bailey, J.S., Frye, J.G., Endale, D.M., Franklin, D.H., Schomberg, H.H., Fisher, D.S., Sharpe, R.R. 2008. Rainfall and tillage effects on transport of fecal bacteria and sex hormones 17ß-estradiol and testosterone from broiler litter applications to a Georgia Piedmont Ultisol. Science of the Total Environment. 403(1-3):154-163.

Cox Jr, N.A., Richardson, L.J., Buhr, R.J., Musgrove, M.T., Berrang, M.E., Bright, W. 2007. Bactericidal effect of several chemicals on hatching eggs inoculated with Salmonella serovar Typhimurium. Journal of Applied Poultry Research. 16:(4)623-627.

Hannah, J.F., Fletcher, D.L., Cox Jr, N.A., Smith, D.P., Cason Jr, J.A., Northcutt, J.K., Buhr, R.J., Richardson, L.J. 2008. Effect of sand and shaking duration on the recovery of aerobic bacteria, coliforms, and Escherichia coli from prechill broiler whole carcass rinsates. Applied Poultry Research. 17:(2)272-277.

Bailey, J.S., Rolon, A., Hofacre, C.L., Holt, P.S., Wilson, J.L., Cosby, D.E., Richardson, L.J., Cox Jr, N.A. 2007. Humoral and mucosal-humoral immune response to a salmonella vaccination program in broiler breeders. International Journal of Poultry Science. 6(3):172-181.

Rolon, A., Bailey, J.S., Hofacre, C.L., Holt, P.S., Wilson, J.L. 2007. Intestinal humoral immune response and resistance to salmonella challenge of progeny from breeders vaccinated with killed antigen. International Journal of Poultry Science. 6(6):417-423.

Bailey, J.S., Rolon, A., Hofacre, C.L., Holt, P.S., Wilson, J.L., Cosby, D.E., Richardson, L.J., Cox Jr, N.A. 2007. Resistance to Challenge of Breeders and Their Progeny with and without Competitive Exclusion Treatment to Salmonella Vaccination Programs in Broiler Breeders. International Journal of Poultry Science. 6(6):386-392.

Northcutt, J.K., Cason Jr, J.A., Ingram, K.D., Smith, D.P., Buhr, R.J., Fletcher, D.L. 2008. Recovery of Bacteria from Broiler Carcasses after Immersion Chilling in Different Volumes of Water, Part2. Poultry Science. 87:573-576.

Cox Jr, N.A., Richardson, L.J., Buhr, R.J., Northcutt, J.K., Bailey, J.S., Cray, P.J., Hiett, K.L. 2007. Recovery of Campylobacter and Salmonella serovars from the spleen, liver and gallbladder, and ceca of six and eight week old commercial broilers. Journal of Applied Poultry Research. 16(4):477-480.

Cason Jr, J.A., Hinton Jr, A., Northcutt, J.K., Buhr, R.J., Ingram, K.D., Smith, D.P., Cox Jr, N.A. 2007. Partioning of external and internal bacteria carried by broiler chickens before processing. Journal of Food Protection. 70:2056-2062.

Cason Jr, J.A., Buhr, R.J., Richardson, L.J., Cox Jr, N.A. 2007. Internal and external carriage of inoculated salmonella in broiler chickens. International Journal of Poultry Science. 6:952-954.

Line, J.E., Bailey, J.S., Berrang, M.E. 2008. Addition of sulfamethoxazole to selective media aids in the recovery of campylobacter spp. from broiler rinses. Journal of Rapid Methods and Automation in Microbiology. 16(1):2-12.

Line, J.E., 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., Siragusa, G.R., Stern, N.J. 2008. Isolation and Purification of Enteroicin E-760 with a Broad Antimicrobial Activity Against Gram-positive and Gram-negative Bacteria (AAC01569-06 Version 3). Antimicrobial Agents and Chemotherapy. 52:1094-1100.

Siragusa, G.R., Haas, G.J., Matthews, P., Smith, R., Buhr, R.J., Macdougald, L., Dale, N. 2008. Humulus lupus Beta-acids Administered Through Water Reduce Clostridium perfringens Challenge Strains in the Chicken Intestinal Tract Midgut and Ceca.. Antimicrobial Agents and Chemotherapy. 61:853-8.

Last Modified: 7/31/2014
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