Impact of Hog and Turkey Farm Production Practices on Molecular Epidemiology of Campylobacter, Salmonella, and Emerging Foodborne Pathogens

APPROACH: Time of entry of Salmonella and Campylobacter will be monitored in turkeys. The study will document flock management practices which affect the prevalence of these foodborne pathogens initially in the brooder period and ultimately throughout live production. Key virulence attributes of C. jejuni and C. coli strains recovered from turkeys will be characterized in vitro (cell invasion assays)and in vivo (day of hatch poult model.) Ultimately, differential gene expression formats will be used to differentiate a virulent from virulent isolates of C. coli and C. jejuni. Bacterial and fungal communities of the ceca of domestic and wild turkeys will be described. This initial survey will provide a measurement of diversity between wild and domestic birds. Analysis of total community flux over time will focus on kinetics of Campylobacter community development and stability in the turkey ceca. Organisms that correlate with Campylobacter colonization or exclusion will be identified. Second generation PCR assays will be developed to detect and quantify Salmonella and Campylobacter on hog and turkey carcasses. Monitoring viscera will serve as an indicator of on-farm versus in-plant sources of contamination. These studies will assist in determining the critical control points of contamination during slaughter. IBC-0281R BSL-2/BSL2N; Recertified 6/30/06. <P>PROGRESS: 2005/10 TO 2006/09<BR>
Progress 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? Food animals are the primary source of bacterial human foodborne pathogens. Following USDA-Food Safety and Inspection Service (FSIS)- Hazard Analysis and Critical Control Points (HACCP) regulation and consumer concerns, livestock producers are attempting to reduce the on- farm prevalence of these pathogens. The newly drafted project has four objectives: (1) Using optimized sampling strategies, enumeration, and molecular diagnostics, identify management practices resulting in high and low Salmonella and Campylobacter prevalence turkey farms and monitor the efficacy of on-farm intervention strategies targeting specific risk factors; (2) Identify key virulence attributes to differentiate avirulent commensal Salmonella and Campylobacter from those pathogenic strains that pose a human public health threat; (3) Develop molecular methods to assess the dynamics of the microbial intestinal flora throughout hog and turkey production; identify microbes associated with gut colonization by, and population shifts of, foodborne pathogens; (4) Determine prevalence and quantities of recognized foodborne pathogens, principally Salmonella but also Campylobacter and Yersinia, in hog carcasses and organs. In the U.S., approximately 5 million cases of bacterial foodborne illness occur annually at a cost of approximately $3.2 billion. The U.S. turkey industry is valued at $7.8 billion with per capita consumption ~ 18 pounds annually. An estimated 458,000 pounds of whole or cut-up turkey parts are exported annually. Thus, international embargoes against U.S. poultry attributed to Salmonella contamination impact the industry. Turkeys are a natural reservoir for C. jejuni, C. coli and Salmonella. The recent ARS Salmonella summit was convened in response to CDCs reports that although E. coli O157:H7, Campylobacter and Listeria- associated enteritis have declined and are approaching Healthy People 2010 goals, Salmonella contamination in poultry is increasing. Salmonella reduction in poultry, meat animals, and produce is now a priority for FSIS and other regulatory agencies. ARS has been advised of the need for data to support hazard evaluation and risk assessment of bacterial pathogens on-farm. Further, in the U.S., over 300,000 swine are slaughtered daily. Approximately 3% to 50% of those animals harbor Salmonella when they reach the kill floor. Hogs are also a major animal reservoir for Yersinia enterocolitica, a bacterial agent which is which is transmitted to humans by pork, is the sixth most frequent cause of hospitalizations ascribed to foodborne illness, and is under FoodNet surveillance. Thus, reducing the prevalence of these bacteria in turkeys and hogs may lower human foodborne illnesses and deaths. These studies are relevant to regulatory agencies as they conduct risk assessments, to the swine and turkey industries, and to the consumers in the global market. Sampling methods for isolation, identification, characterization, and quantification of foodborne pathogens in livestock are major research priorities for ARS pre-harvest food safety efforts. Project Plan focuses on understanding the ecology and epidemiology of foodborne pathogens in swine and turkey production processes under Sections 1.1.2 (Epidemiology); 1.1.3 (Ecology, host pathogens and chemical residue relationships); and 1.2.3 (Production and processing ecology), improved sampling methods under Section 1.1.1 (Methodology); and recommendations for on-farm production practices under Section 1.1.4 (Intervention Strategies). This research is central to achieving effective intervention strategies to reduce on-farm transmission (Action Plan Priority Objective 1.4.1). In- house efforts are complemented by access to farms and processing plants facilitated by the Iowa Turkey Federation and extramural collaborations. <p>2. List by year the currently approved milestones (indicators of research progress)<BR> Year 1 (FY 2006) <BR>Objective 1: Using optimized sampling strategies, enumeration, and molecular diagnostics, identify management practices resulting in high and low Salmonella and Campylobacter prevalence turkey farms and monitor the efficacy of on-farm intervention strategies targeting specific risk factors. Subobjective 1.1 Begin flock sampling. Subobjective 1.2 With FSIS design questionnaire. <BR>Objective 2-Identify key virulence attritubes to differentiate Salmonella and Campylobacter avirulent commensals from those pathogenic strains that posse a public health threat in humans. Subobjective 2.1 Map Campylobacter in crop, intestine, ceca. Subobjective 2.4 Recruit 0.5 SY. Objective 3- Develop molecular methods to assess the dynamics of the microbial intestinal flora throughout hog and turkey production. Identify microbes associated with gut colonization by, and population shifts of, foodborne pathogens. Subobjective 3.1 Begin bacterial, fungal OFRG. Subobjective 3.2 Complete on-farm flock data collection. Objective 4-Determine prevalence and quantities of recognized foodborne pathogens, principally Salmonella but also Campylobacter and Yersinia in hog carcasses and organs. Subobjective 4.1 Evaluate single tube Salmonella MPN. <BR>Year 2 (FY 2007) <BR>Objective 1 Subobjective 1.1 - Evaluate data; redesign sampling strategy, if needed. Subobjective 1.2 - With risk assessors analyze questionnaire. Objective 2 Subobjective 2.2 - Begin in vivo virulence assays with C. coli. Subobjective 2.2 - Begin in vitro virulence assays with C. coli. Subobjective 2.4 Begin C. coli microarray analysis. Objective 3 Subobjective 3.1 - Complete bacterial, fungal OFRG. Subobjective 3.2 - Complete analysis. Subobjective 3.3 - Begin antibiotic dissection of gut community. Objective 4 Subobjective 4.1 - Evaluate DNA extraction protocols. Subobjective 4.2 - Evaluate DNA and real-time PCR forms with various organs. Subobjective 4.3 - Plan studies with FSIS; secure plant access. <BR>Year 3 FY 2008 <BR>Objective 1 Subobjective 1.1 - Data analysis; retest if needed. Subobjective 1.2 - Present data and survey responses to risk assessors. Objective 2 Subobjective 2.2 - Continue screening in vivo models. Subobjective 2.3 - Continue screening cell lines. Subobjective 2.4 Select genes for quantitative PCR. Objective 3 Subobjective 3.1 - Present and publish results. Subobjective 3.3 - OFRG and validation. Objective 4 Subobjective 4.1 - Evaluate DNA extraction protocols. Subobjective 4.3 - Begin in-plant sampling. <BR>Year 4 FY 2009 <BR>Objective 1 Subobjective 1.1. - Present results. Objective 2 Subobjective 2.2 Analyze data. Subobjective 2.3 - Analyze data. Subobjective 2.4 Select genes for quantitative PCR. Objective 3 Subobjective 3.3 Examine naturally occurring gradients. Objective 4 Subobjective 4.1 - Evaluate real time PCR formats. Sobobjective 4.3 - Continue in-plant sampling. <BR>Year 5 FY 2010 <BR>Objective 1 Subobjective 1.1 Publish results. <BR>Objective 2 Subobjective 2.2 Publish results. Subobjective 2.3 Publish results.<BR> Objective 3 Subobjective 3.3 Isolation of microbes. <BR>Objective 4 Subobjective 4.2 Publish data. Subobjective 4.3 Complete plant sampling; analyze data. 4a List the single most significant research accomplishment during FY 2006. This accomplishment focuses on understanding the ecology and epidemiology of foodborne pathogens in swine and turkey production processes under Sections 1.1.2 (Epidemiology); 1.1.3 (Ecology, host pathogens and chemical residue relationships); and 1.2.3 (Production and processing ecology). Total analysis of intestinal bacteria communities: We have shown that intestinal bacterial communities differ between domestic and wild turkeys; that significant community differences emerge during the life of a turkey from day of hatch to market-weight; that intestinal communities may be flock-specific; and that intestinal communities of non-transported and transported turkeys differ. Description of the intestinal microbiota offers the potential to determine the molecular basis for colonization of turkeys with Camplyobacter and Salmonella. 4b List other significant research accomplishment(s), if any. These accomplishments focus on improved sampling methods under Section 1. 1.1 (Methodology); and recommendations for on-farm production practices under Section 1.1.4 (Intervention Strategies) and address effective intervention strategies to reduce on-farm transmission (Action Plan Priority Objective 1.4.1). In-house efforts are complemented by access to farms and processing plants facilitated by the Iowa Turkey Federation and extramural collaborations. We screened for Campylobacter in three types of egg layer housing systems: a) buildings in which hens are confined but kept on the ground (n=4 flocks; non-caged birds); b) high-rise buildings with chickens in cages and a manure pit beneath the chickens (n=4 flocks) and c) buildings with chickens in cages and a manure belt beneath the chickens (n=4 flocks) in collaboration with Iowa State University (Xin et al). When the three housing types were compared, the prevalence of Campylobacter spp., especially C. coli, was significantly higher in non-caged birds which were confined but kept on the ground (p<0.05). No such differences were seen among flocks in the distribution of either C. jejuni or in Salmonella. This indicates that the consequences of management practices should include consideration of foodborne pathogens. We have miniaturized Salmonella isolation and detection into a microtiter deep-well format. Validation studies with field samples of turkey ceca and hog feces in side-by-side comparisons with traditional conventional isolation have shown agreement between the two assays. The microtiter format has the potential of streamlining detection of Salmonella. <BR>5. Describe the major accomplishments to date and their predicted or actual impact. This accomplishment focuses on understanding the ecology and epidemiology of foodborne pathogens in swine and turkey production processes under Sections 1.1.2 (Epidemiology); 1.1.3 (Ecology, host pathogens and chemical residue relationships); and 1.2.3 (Production and processing ecology). Total analysis of intestinal bacteria communities: Molecular analysis of bacteria and fungi from livestock intestines begins with rigorous testing of DNA extraction protocols and selection of methods to avoid bias inherent in template preparation. Using these optimized methods, we have subsequently shown that intestinal bacterial communities differ between domestic and wild turkeys; that significant community differences emerge during the life of a turkey from day of hatch to market-weight; that intestinal communities may be flock-specific; and that intestinal communities of non-transported and transported turkeys differ. These accomplishments focus on improved sampling methods under Section 1. 1.1 (Methodology); and recommendations for on-farm production practices under Section 1.1.4 (Intervention Strategies) and address effective intervention strategies to reduce on-farm transmission (Action Plan Priority Objective 1.4.1). In-house efforts are complemented by access to farms and processing plants facilitated by the Iowa Turkey Federation and extramural collaborations. We screened for Campylobacter in three types of egg layer housing systems: a) buildings in which hens are confined but kept on the ground (n=4 flocks; non-caged birds); b) high-rise buildings with chickens in cages and a manure pit beneath the chickens (n=4 flocks) and c) buildings with chickens in cages and a manure belt beneath the chickens (n=4 flocks) in collaboration with Iowa State University (Xin et al). When the three housing types were compared, the prevalence of Campylobacter spp., especially C. coli, was significantly higher in non-caged birds which were confined but kept on the ground (p<0.05). No such differences were seen among flocks in the distribution of either C. jejuni or in Salmonella. This indicates that the consequences of management practices should include consideration of foodborne pathogens. We have miniaturized Salmonella isolation and detection into a microtiter deep-well format. Validation studies with field samples of turkey ceca and hog feces in side-by-side comparisons with traditional conventional isolation have shown agreement between the two assays. The microtiter format has the potential of streamlining detection of Salmonella. <BR>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? Fundamental knowledge on the kinetics of the microbiota in the turkey ceca is critical in understanding the impact of on-farm interventions, including administration of pre- and probiotics. Basic knowledge on the dynamics of cecal microbiota was emphasized as a research need by the Iowa Turkey Federation at the Iowa State University-USDA Poultry Science Day. Details of the effect of feed withdrawal, transport, and holding on the intestinal populations of Campylobacter jejuni and C. coli in the market weight turkey have been published and presented to various user groups, including attendees at the annual meeting of the National Turkey Federation, Iowa State University-USDA Poultry Science Day, and the ARS Salmonella summit. Similarly, the lack of effect of perimarketing events on Salmonella shedding in turkeys in contrast to what occurs in market weight hogs is being presented to regulatory, scientific, and commodity groups. Despite the FSIS emphasis on Salmonella reduction, although specific on-farm interventions are well known, the economics of such undertaking may be cost prohibitive. The finding of higher levels of Campylobacter in non-caged hens indicates that the consequences of management practices should include consideration of foodborne pathogens. These findings will be available to commodity groups following publication of these results in the scientific literature. The microtiter format has the potential of streamlining detection and ultimately enumeration of Salmonella. Rapid screening of livestock may be critical in evaluating on-farm Salmonella reduction strategies. These findings will be available to commodity groups following publication of these results in the scientific literature. <BR>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). Presented, Report on the National Advisory Committee on Microbiological Criteria for Foods. Annual meeting of the National Turkey Federation in Orlando, Florida. February 14, 2006. Presented, Turkey Trips dont aggravate contamination. Tristate Food Safety Consortium Spring Newsletter. 2006. Presented, Do feed withdrawal, livehaul and holding influence the prevalence of Salmonella and Campylobacter in turkeys. Annual meeting of the National Turkey Federation, in Orlando, Florida. February 14, 2006.