2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? The Centers for Disease Control and Prevention estimates that Campylobacter spp., Salmonella spp. (non-typhoidal), and Clostridium perfringens are the three leading bacterial etiologies of human food-borne illness in the United States, with approximately 2.5 million, 1.4 million, and 248.5 thousand cases per year, respectively. Additionally, these three bacteria are responsible for approximately 30,000 hospitalizations and 700 deaths each year, costing 2 to 5 billion dollars annually. While these bacteria are associated with a variety of foods, the handling and consumption of poultry or poultry products are considered the primary source for bacterial food-borne disease in humans. Another bacterial pathogen closely associated with the consumption of poultry is Listeria monocytogenes. While L. monocytogenes accounts for only 2% of all food related illnesses in the Unites States per year, it is responsible for 28% of all food related deaths. The pathways and mechanisms (gastrointestinal/reproductive tract microbial ecology, adhesions, toxins, antigenic phase variation, biofilms, etc) involved in contamination of poultry flocks by these pathogens are not well defined, therefore, effective intervention strategies are lacking. It is imperative to understand the molecular factors involved in the colonization of poultry and how the microbial ecology of the chicken gastrointestinal system will be affected by the presence or absence of antibiotics in feed. We propose to conduct in depth investigations to (1) characterize molecularly (using DNA sequence and rep-PCR analysis) selected isolates of Campylobacter spp., Salmonella spp, and Clostridium perfringens to improve identification of critical factors for pathogen contamination of poultry; (2) identify (using suppressive subtractive hybridization and gene expression analysis facilitated by microarray technology) salient microbial and host factors that promote colonization of these pathogens in the chicken host; and (3) determine (culturally, fluorescently, and molecularly) the microbial ecology of the chicken gastrointestinal tract, the chicken reproductive tract, chicken internal organs, and exogenous biofilms. The identification of the critical sources and mechanisms of Campylobacter spp. contamination in poultry is important to allow for the development of novel intervention strategies. These interventions will facilitate the delivery of pathogen free birds to the abattoir, and consequently, should reduce the incidence of human exposure. This research is an important component of National Program 108 - Food Safety (animal and plant products). The mission of this NP is to decrease the hazards of both introduced and naturally occurring toxicants in food and feed, including pathogenic bacteria. This CRIS addresses the pre-harvest pathogen component of the national program. 2.List by year the currently approved milestones (indicators of research progress) Year 1 (FY2002) 1. Identify the most critical sources of Campylobacter spp. contamination; 2. Develop methods for the rapid recovery and detection of Campylobacter spp. in complex matrices. Year 2 (FY2003) 1. Assess the interactions of poultry intestinal tract organisms with Campylobacter spp. Year 3 (FY2004) 1. Continue identification of the most critical sources of Campylobacter spp. contamination. 2. Continue assessing the interactions of poultry intestinal tract organisms with Campylobacter spp. Year 4 (FY2005) 1. Provide predictive models (quantitative) risk assessment. 2. Create directed interventions aimed at reducing levels of Campylobacter spp. Year 5 (FY2006) 1. Complete identification of most critical sources of Campylobacter spp. contamination. 2. Complete predictive models (quantitative) risk assessments. 3. Complete molecular epidemiology investigations utilizing rep-PCR. 4a.List the single most significant research accomplishment during FY 2006. Bacteriocin treatment of chickens by feeding consistently reduced C. jejuni levels in their gastrointestinal system by at least one-million fold as compared with levels found in untreated birds. Anti-Campylobacter jejuni activity was evaluated among greater than 1,200 isolates of different lactic acid bacteria and Lactobacillus salivarius strain NRRL B-30514 was identified as an inhibitor of C. jejuni replication. The anti-bacterial peptide, bacteriocin OR-7, was purified from the L. salivarius strain and the amino acid sequence was consistent with Class IIa bacteriocins. Previously identified bacteriocins from L. acidophilus had activity only to Gram-positive bacteria, whereas OR-7 had activity to the Gram-negative bacterium C. jejuni. This research is a part of National Program 108 - Food Safety (animal and plant products) and addresses the reduction of bacterial pathogens in the food supply. 4b.List other significant research accomplishment(s), if any. Disinfectant activity of a quaternary ammonium compound with hydroperoxide ion against L. monocytogenes reduced bacterial counts. Technique-sensitive methods provided quantitative data for comparison of disinfectant activity against L. monocytogenes isolates from the poultry environment for this compound. Novel indicators to monitor Campylobacter spp. growth were investigated through a letter of confidentiality and material transfer agreement with R&F Labs. A series of 13 experiments were conducted in search of possible indicators to be utilized in campylobacter selective media to impart a specific color change to the media upon growth of Campylobacter spp. colonies. This is a difficult task given the non-fermentative nature of campylobacter. A C. jejuni flagellum-specific phage PV22 from Proteus vulgaris was identified in sewage drainage. This phage interacted with C. jejuni by attachment to flagella followed by translocation of the phage to the polar region of the bacterium. Electron microscopic examination revealed adsorption of PV22 on C. jejuni flagella. There may be two interesting applications of this effect. First, it may be possible to test phage PV22 as an antimicrobial agent to decrease C. jejuni colonization of the chicken intestine. Second, the phage could potentially be utilized for investigating biogenesis of C. jejuni flagella. Acidifying litter treatments caused a delay in onset of Campylobacter colonization among broiler chicks while Salmonella levels remained unaffected by acidification treatment. Campylobacter populations and Salmonella incidence associated with unprocessed, whole carcass rinse samples analyzed at the end of production were unaffected by litter acid treatment. Significantly more Campylobacter spp. were recovered by a newly developed biphasic culture technique. Combining the enumerative capabilities of a microtiter dilution well assay with biphasic growth conditions was better than traditional plating methods. Reduction of relative humidity in poultry rearing houses through ventilation and water handling practices that promote dryer litter lowered Salmonella contamination during poultry production. The results of these studies also suggest that these practices should help reduce exposure of poultry to viable Campylobacter in litter. Bacteriocin peptide (LW118) from Lactobacillus salivarius reduced C. perfringens populations in experimentally-challenged broiler chickens. Also, previously reported bacteriocin S-760 was found to inhibit replication of pathogenic bacteria such as C. difficile and Enterobacter sakazaki as well as Campylobacter. 4c.List significant activities that support special target populations. In cooperation with the Centers for Disease Control (CDC) a project was initiated to determine Campylobacter populations in retail chicken breast samples. Initial studies were completed to select methods for recovery of campylobacters from raw chicken. Research on drug-free chicken broiler rearing problems supports the rapidly enlarging sector of the U.S. industry rearing broiler chickens without antibiotic growth promoters. The collaboration between PMSRU, ARS, USDA and the State Research Center for Applied Microbiology and Biotechnology (Obolensk, Russian Federation) also continues to provide support for former biological weapons scientists. 4d.Progress report. Our research involving isolating Clostridium perfringens and Campylobacter jejuni microbial antagonists (direct fed anti-microbial botanicals, lytic phage and bacteriocin peptides) continues to make significant progress. This work is at the point of patent application submission and publication and rapidly approaching the stages of scale-up and production consideration followed by licensure. We have been successful in forming a relationship with a U.S.-based broiler company located in Russia which will collaborate with us in field testing the bacteriocin strategy for food-borne pathogen reduction among commercial poultry. The research has been expanded to include reduction of other pathogens such as Salmonella spp. which has been demonstrated to be effective. 5.Describe the major accomplishments to date and their predicted or actual impact. We continue to identify new bacterial isolates antagonistic to Campylobacter jejuni and other food-borne bacteria such as Salmonella spp. and Clostridium perfringens, to biochemically characterize the associated antimicrobial peptides and make production of these more efficient. The US State Department funded through their Biological Industry Initiative (BII) a project to produce large quantities of selected bacteriocins. Commercial production-scale field trials employing one-half million broiler chickens with Russian collaborators at the USA Poultry and Egg Export Council-sponsored poultry production facility in Russia will be initiated as a result of this agreement. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? 0175.02 Serial Number 10/426,688 filed 05/01/2003 was issued as U.S. Patent No. 6,989,370 on 01/24/2006, "Bacteriocins and Novel Bacterial Strains." A patent describing the Paenibacillus organism producing the antimicrobial protein (bacteriocin) and its application to successfully reduce one-million fold Campylobacter jejuni in the live chicken. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). International Partnership for Poultry Safety. Agricultural Research Service Magazine. November, 2005. Review Publications Zhilenkov, E.L., Popova, V.M., Popov, D.V., Zavalsky, L.Y., Svetoch, E.A., Stern, N.J., Seal, B.S. 2006. The ability of flagellum-specific proteus vulgaris bacteriophage pv22 to interact with campylobacter jejuni flagella in culture. Virology Journal. 3:50-54. Line, J.E. 2006. Influence Of Relative Humidity On C. Jejuni Transmission in Broiler Chickens. Poultry Science. 85:1145-1150. Line, J.E., Siragusa, G.R. 2006. Biphasic microtiter methods for campylobacter recovery and enumeration. Journal of Rapid Methods and Automation in Microbiology. 14:182-188. Line, J.E. 2006. Comparison of enrichment vessels for recovery of campylobacter spp. from broiler rinse samples. Journal of Rapid Methods and Automation in Microbiology. 14:110-117. Svetoch, E.A., Eruslanov, B.V., Pokhilenko, V.D., Kovalev, Y.N., Volodina, L.I., Perelygin, V.V., Mitsevich, E.V., Mitsevich, I.P., Borzenkov, V.N., Levchuk, V.P., Svetoch, O.E., Kudriavtseva, T.Y., Stern, N.J. 2006. Control of campylobacter jejuni colonization in chickens treated with probiotics or bacteriocins [abstract]. Fifth Joint RRI-INRA Gastrointestinal Tract Microbiology Symposium. 45(Suppl 1):S32:62.