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I. E-probe Diagnostic Nucleic Acid Analysis (EDNA). 1. Test massively parallel sequencing (MPS) diagnostics on plants infected with plant pathogens. 2. Test MPS forensic capacity on selected plant pathogens. 3. Test MPS ability to detect pathogens genetically engineered to express toxins or proteins. <P>II. Develop and validate a forensic strategy for a model plant disease. 1. Devise an assay to discriminate Fusarium proliferatum (Fp) strains for investigation of strain origin. 2. Test the assay in a real-world incident. 3. Develop and validate a real time Fp PCR assay. <P>III. Determine the impact of elapsed time on the validity of molecular forensic typing. 1. Evaluate rates of evolutionary change in Psuedomonas syringae pv. tomato, and their impacts on forensics investigations. <P>IV. Identify bacterial characteristics that can reveal a history of laboratory culture. 1. Identify detectable signatures of laboratory culture. <P>V. Characterize Salmonella colonization of cantaloupe. 1. Determine if S. enterica can internalize and move systemically in cantaloupe when introduced through wounds or flowers. 2. Test the effect of other plant resident bacteria on S. enterica behavior on cantaloupe rind. 3. Test insects from Oklahoma cantaloupe fields for the presence of human enteric pathogens. <P>VI. Effect of fumigant use in tree nut orchards on shell surface microbial communities. 1. Determine microbial load on walnuts and almonds harvested from trees treated, or not, with fumigants for plant pathogen control. 2. Determine the populations of Salmonella spp. and E. coli O157:H7 on walnuts and almonds from trees treated, or not, with fumigants. <P>VII. Enhance detection technology for forensic analysis of foodborne Salmonella and E. coli O157:H7 on fresh produce. 1. Enhance PCR detection of Salmonella and E. coli O157:H7 by developing and testing several strategies for increasing sensitivity and specificity of PCR primers.
Non-Technical Summary:<br/>
In 2004, Homeland Security Presidential Directive 9 mandated a national policy to defend U.S. agriculture and food systems against terrorist attacks, major disasters, and other emergencies. HSPD-9 found that U.S. agriculture and food systems are vulnerable to diseases and pests that occur naturally, unintentionally, or are intentionally delivered by acts of terrorism/biocrime. Agriculture plays a critical role in our nation's infrastructure, gross domestic product, trade and employment. Plant diseases cause severe economic repercussions; diseases of US crops cost billions of dollars/year. Consequences include costs of disease management, higher food prices, reduced quality and availability, trade disruptions, human and animal health costs, and loss of consumer confidence. Preparedness requires a strong national security plan that includes microbial forensics and criminal attribution. Forensic strategies must include (a) assuring high stringency; (b) tracing pathogen origin and movement; (c) timing and site of introduction; (d) perpetrator identification; (e) evidence for criminal attribution; and (f) links to law enforcement and security communities. Features critical to a robust capability in microbial forensics include: (1) stringent methods for accurate, repeatable and highly reliable comparison and validation of microbial forensic methods. (2) sampling methods that consider sample size and quality, variation among crops, sampling sites and organisms, preserving sample integrity, documentation and security; (3) knowledge of genome dynamics, phylogenetics and systematics of pathogens that are influenced by the environment, hosts and other microbes; (4) molecular markers that are immutable, discriminatory and diagnostic even under the influence of mutation, evolution and environment; (5) consideration of ecology and background clutter, especially with respect to epidemiological tools such as climate matching, trajectory analysis and reconstructive tools; (6) understanding of post-translational modifications; (7) standard, validated discrimination and match criteria; and (8) integrated bioinformatics, data analysis and computational strategies for bioforensics. OSU's National Institute for Microbial Forensics & Food and Agricultural Biosecurity, established in 2007 in response to the needs indicated above, is a multidisciplinary team of OSU faculty experienced in multiple facets of plant agriculture, food security, and forensic science. Its mission is to build on and enhance existing programs to address issues of crop and food biosecurity, and their impacts on people and economies. Its goals are to (a) assess current capabilities in forensics as related to plant pathogens and food safety; (b) provide strategic planning, long-range vision and prioritization of needs in this emerging field; (3) foster cooperative research on crop and food safety forensics issues; (4) serve as a focal point for communication, collaboration, funding initiatives, and outreach; (5) develop education and training opportunities; (6) communicate and work in parallel with forensics programs for animal and human pathogens.
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Approach:<br/>
I. 1. a) Generate sample sequence databases (SSDs) for diseased plants using 454 MPS technology, b) Use SSDs to evaluate pathogen query sequence selection methods, c) Test the diagnostic capacity of MPS and pathogen diagnostic sequences using blinded panels. 2. a) Use the SSDs to evaluate forensic typing query sequence selection methods, b) Test the forensic capacity of MPS and the PFTSs using blinded panels. 3. a) Generate SSDs for plant samples, some spiked with toxin genes or genetic engineering sequences, b) Test the capacity of MPS to detect hallmarks of genetically engineered pathogens using blinded panels.
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II. 1: a) Assess multi-locus sequence typing (MLST) and single sequence repeats (SSR), for their suitability in discriminating Fp strains. b) Validate and standardize the assay for forensic level stringency. 2: a) Test H: Fp strains from onion sets are genetically similar to those from the seeds planted to produce them. Fp will be isolated from onion sets/ seeds and DNA extracted. Isolate relatedness will be assessed with MLST or SSR and phylogeny software. b) Test H: Fp isolates from onion bulbs in production fields are genetically different from background Fp collected nearby. Fp isolates from onion bulbs (production field) will be compared as above with Fp from adjacent vegetation and soil. 3: Develop/validate a Fp real time PCR assay. a) Primers will target the translation elongation factor 1? (EF1?) gene. b) Validation panels: (1) near-neighbors and outgroups, (2) agriculturally significant plants, (3) agriculturally significant animals.
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III. a) Determine the stability of VNTR loci in the Pst genome; b) Determine rates of evolution of Pst housekeeping genes; c) Assess the impacts of these factors on the suitability of VNTR and MLST typing for Pst forensic analysis.
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IV. a) DNA from sequential passages will be assessed by MLST and MLVA for the rates of evolutionary change of core genome loci; b) Amplicon sequences will be aligned and assessed for evolutionary changes; c) At intervals, bacteria will be assessed for phenotypic changes.
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V. 1. Test whether Salmonella can colonize cantaloupe rinds. 2. a) Assess S. enterica's ability to form biofilms on cantaloupe surfaces and enter the plant. b) Test the effect on colonization of the presence of other microbes. 3. Screen insects from OK cantaloupe production fields for Salmonella.
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VI. 1. Assess total microbial load by washing nuts and plating serially diluted wash buffer. Place recovered microbes into general taxonomic groups. 2. Assess titers of human pathogens by plating aliquots of wash buffer onto selective media.
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VII. Assess the impact, on PCR sensitivity and specificity, of adding 5' tags to PCR primers for foodborne pathogens, a method that has produced several-fold sensitivity enhancement for other microbes.