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The goal of this project is to optimize the application of sodium chlorate and sodium nitrate to reduce Salmonella shedding in growing pigs. We will achieve this goal by measuring the response of Salmonella to differing chlorate dosing strategies in a series of strategically designed in vitro and in vivo models, with and without the addition of sodium nitrate and nitrate. <P> Objectives of project are to: <OL> <LI> Define the chlorate, nitrate and pH levels reached in GI tract of pigs following doses that have successfully reduced Salmonella CFU / g in prior live animal research. <LI> Define the effects of chlorate dose and supplemental nitrate on Salmonella survival using in vitro models of Salmonella infection under aerobic, anaerobic and microaerophilic conditions. <LI> Define optimum chlorate / nitrate dosing protocols, including dose level, dose frequency, and dose duration in weaning and market age pigs. <LI>Describe the effect of dosing regimens on Salmonella shedding and harborage in live pigs, including the duration of effect after administration are discontinued. </OL> Outputs of the project are: <OL> <LI>Findings of experiments addressing each of the four objectives. We will assess data quality during each phase of the project experiments, design, implementation, data collation, and analysis. <LI> Presentation of findings at scientific conferences. <LI> Scientific manuscripts published in peer reviewed journals. <LI>A trained graduate student, included on authorship at presentations and in scientific papers. </OL> The target dates for conduct of experiments and development of outputs is as follows: a) Objective 1, Define chlorate and nitrate in live animals, start January 2008, end July 2008. b) Objective 2, Define in vitro effective concentrations for chlorate and nitrate, start August 2008, end April 2009. i) Submit and present at scientific conference for objectives 1-2 by July 2008. ii) Submit manuscripts for objectives one and two for peer reviewed publication by September 2008. c) Objective 3: Live animal experiments, microbial data, nitrate assays and initial chlorate assays, start May 2009, end October 2009. i) Chlorate assays, in vitro chlorate / nitrate optimal dose and duration defined, start June 2009, complete by December 2009. ii) January 2010, submit and present at scientific conference. By January 2010 submit manuscript for peer reviewed publication. iii) Objective 4: Experiments start December 2009. 1) In December of 2010, begin preparation of manuscript; submit for peer reviewed publication January 2011.
NON-TECHNICAL SUMMARY: Salmonella enterica in food producing animals provides a food safety risk for humans, with 1.4 million human illness and more than 500 deaths per year in the U.S, according to one estimate. Although the USDA Food Safety Inspection Service has regulated Salmonella on carcasses since 1997, there is no official program to regulate Salmonella on farms. One critical reason that intervention to reduce Salmonella on swine farms has not been realized is that there are currently not interventions that have been proven to reliably and economically result in reduction of Salmonella in pigs. Our labs have demonstrated that the application of sodium chlorate / nitrate can be an effective means to reduce Salmonella shedding and / or carriage in food producing animals that have been experimentally and naturally challenged with Salmonella. This beneficial effect can persist for at least 10-days after discontinuation of therapy. However, critical questions remain unanswered regarding 1) the minimal effect dose and duration, 2) the need for and/or necessary dose of nitrate, and 3) the effective duration of beneficial effects after the compounds are withdrawn. If unanswered, these questions will prevent the development and application of this promising food safety tool. The goal of this study is to optimize the application of sodium chlorate to reduce Salmonella shedding in swine. We will initially test a dose range of chlorate and nitrate used in prior live animal models to establish a range of potentially effective target concentrations for further testing. We will then use laboratory bench top (in vitro) models to narrow the range of concentrations to be evaluated in the live animals. The dose and duration of chlorate administration required to achieve these gut concentrations will be evaluated in live pigs. Samples of gut (cecal) contents will be collected through surgically placed indwelling tubes (cannula). Chlorate and nitrate concentrations will be measured using the latest, most effect detection techniques, including mass spectrometry for chlorate. Dose and duration combinations identified by this work will then be assessed for effectiveness in killing Salmonella in growing pigs which have been given live Salmonella orally. The findings of work in this project will be communicated to the scientific community at meetings and by publication in peer reviewed scientific journals. We will seek critique at the University of Wisconsin-Madison and at the USDA Food Safety Laboratory (College Station, TX) through graduate student program development through established review processes. We will promote dissemination of findings to other potential end users by listing publications on university and government web pages designed for that purpose. We will make ourselves available to explain our findings and potential implications expected users. We expect that the results of these studies will provide sound data on which to base strategies for the reduction of Salmonella shedding in swine using sodium chlorate and sodium nitrate, and thus advance pork safety and promote public health.
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APPROACH: Live animal experiments will be conducted as approved by the University of Wisconsin-Madison Institutional Animal Use Committee. When assignment of animals to treatments is systematic, the purpose will be to reduce inter-subject variation, and the system will be included in the statistical analysis. Animal subjects will not enter the food supply. Chlorate will be detected using methods established in the USDA ARS Biosciences Research Laboratory. Nitrate will be quantified using liquid chromatography mass spectroscopy at the Wisconsin (State) Veterinary Diagnostic Laboratory (Madison, WI). Test tube (in vitro) models will include defined Salmonella strains, including S. Typhimurium, (NAR-NVSL 95-1776). Bacterial enumeration will be accomplished by serial dilution plating of samples, where pure culture is expected; for mixed culture samples including feces, Most Probable Number techniques will be applied. Anaerobic experiments will use modifications of classical ruminent techniques; modifications have been successfully applied in the laboratory of the Co-PI. For objective 3, pigs will be fitted with cecal cannulae using published methods. For objective 4, animal subjects will be selected from farms with low (<5%) detected Salmonella fecal. The treatment doses and durations tested will be those most promising candidates identified in the prior objectives. We will quantify shedding CFU/g feces by direct plating on media containing naladixic acid and novobiocin, since the challenge strain is resistant to these antimicrobials. Data for all experiments will be checked for quality during and after conduct of the laboratory work, collated using commercial computer software. Analysis will be conducted using commercial statistical software, such as SAS STAT, Data will be interpreted in light of prior knowledge, especially that conveyed in peer reviewed scientific literature. For maximum impact we will 1) assure that the work is properly conducted, 2) present findings at scientific conferences, 3) submit finds to peer review prior to journal publication, and 4) act as a resource for scientists, veterinarians, animal production specialists and others in allied groups to interpret our findings. We will evaluate the impact of our work through feedback rough direct audience feedback by correspondence and other communication, and by tracking citations to our work in other scientific and technical publications. Although not a part of this project, we will assess applications of our work as they develop through our roles as university or research laboratory faculty, clinical swine veterinary work (Bahnson). We will evaluate the impact of our work on our target audiences through feedback received after presentation of outcomes at scientific conferences, through direct audience feedback by correspondence and other communication, and by tracking citations to our work in other scientific and technical publications. Although not a part of this project, we expect to assess applications of our work as they develop through our roles as university or research laboratory faculty, clinical swine veterinary work (Bahnson).