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<p>Focus on emerging diseases: We will identify, characterize and develop improved detection and prevention methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of food animals.
<br>Focus on preventions and interventions: We will develop and improve preventative measures and interventions to reduce the incidence and prevalence of infections of food animals with enteric and foodborne and waterborne pathogens.
<br>Focus on disseminating knowledge: We will provide training or continuing education to disseminate new information to students, producers, veterinarians, diagnostic labs and others to implement interventions and preventative measures.
<br>Group interaction: The group will interact in a variety of ways to facilitate progress including direct collaborations with joint publications, sharing of resources (pathogen strains, gene sequences, statistical analysis, bioinformatics information/expertise), and friendly feedback and facilitation for all research efforts at annual meetings. </p>
<p>NON-TECHNICAL SUMMARY: The long-term goal of this collaborative project is to prevent and control enteric diseases of cattle, swine and chickens with a mandate to decrease food and waterborne illness in the USA. Foodborne illness has been a prominent public health concern for over two decades yet the Centers for Disease Control (CDC) still list many enteric foodborne pathogens as leading causes of morbidity and mortality in the US. Despite many concerted efforts to use hygiene and sanitation measures to control these pathogens in food animals pre- and postharvest, incidence of many food and waterborne pathogens remains high and some are increasing. Nevertheless, a broad range of educational, scientific and practical controls have succeeded in decreasing the incidence of five key foodborne pathogens. Over the last 10 years, our enteric diseases group has been a part of that effort and we are dedicated to prevent and control animal and human disease due to enteric pathogens. Our collaborative efforts harmonize with national efforts established this year under the FDA Food Safety Modernization Act to ensure the US food supply is safe by shifting the focus of federal regulators from responding to contamination to preventing it. A main avenue for prevention is decreasing carriage and disease due to enteric pathogens in food animals. The research group at the University of Wyoming will focus on the development of high throughput identification and typing methods for antimicrobial resistant (AMR) bacteria from cattle operations and wildlife, methods for capture of viruses from bioaerosols in agricultural environments, and development of field-based and rapid diagnostics for microbial pathogens. </p>
<p>APPROACH: We will 1) develop MALDI-TOF MS protocols for identification of antimicrobial resistant bacteria from several cattle feedlots, avian and mammalian wildilife, and agricultural environments using formic acid/ethanol extractions for sample preparation, and 2) examine prevalence of these AMR bacteria to identify potential intervention practices. We will evaluate a multitude of sample preparation techniques to aid in enhancing subtyping capabilities of the developed method. Solvent systems, detergent extractions, enzymatic treatments, sample fractionation, and multiple MALDI matrices will be evaluated using panels of well characterized bacteria from environmental, human, and food sources, with specific focus on E. coli, Enterococcus spp., and Staphylococcus spp. Statistical tools (multivariate analysis and hierarchical and correlational clustering) within the Bruker Daltonics Biotyper software package will then be used for mass spectral comparisons of the isolates to evaluate isolate-to-isolate similarity. Representative panels of the confirmed isolates will also be subjected to pulsed-field gel electrophoresis (PFGE) and antimicrobial sensitivity testing for comparative evaluations of MALDI-TOF MS typing efficacy. The discriminatory mass spectral data, determined through extensive bioinformatic interpretations and by indirect comparisons to PFGE/antimicrobial sensitivity profiles, will be used to build reference mass spectral libraries. To understand the epidemiology of the AMR bacteria, we will examine prevalence via analysis of isolates originating from cattle feces, feed, water, as well as mammalian and avian wildlife then analyze the isolates via PFGE, 16s sequencing, antimicrobial susceptibility tests and compare them to MALDI-TOF MS results to elucidate the epidemiology of these isolates. In addition, we will also develop 3) an enhanced method for capture and concentration of viruses from agricultural environments for subsequent molecular detection which builds upon already existing bioaerosol sampling equipment with modification aimed at increasing sensitivity and simplicity of the procedure in a streamlined process. Laboratory studies will evaluate the modified devices' ability to capture viruses (including multiple variants of influenza viruses) using a bioaerosol chamber. The laboratory evaluations will also determine the minimal concentrations of virus that can be detected in bioaerosols with this method. qRT-PCR will then be used for detection/quantification. Following the laboratory tests, evaluations of the best performing bioaerosol sampling device will be conducted in animal facilities. In addition to RT-PCR testing for the viruses in animal facilities, we will utilize next-generation sequencing to provide a pilot assessment of the total microbiota captured with the bioaerosol samplers, as our proposed method should capture a wide array of microorganisms of relevance to the livestock industry. The work will be performed on bacterial isolates and viral particles and not on live animals, thus not requiring IACUC approval. </p>