Progress 04/15/18 to 04/14/23
Outputs
Target Audience:This Food-Path surveillance project is designed to detect potential human and animal health threats in the entirety of our food production system (from farm to table). As such, our target audience is broad. Our main goal is to develop a tool for public health and food/water safety agencies to use proactively in the continuous monitoring of the food system for potential health threats. Changes/Problems:The most significant issue we've experienced with this project is our significant pivot to respond to the COVID-19 pandemic. As with other laboratories, institutes, and agencies, the pandemic assumed much of our time, efforts, and resources. In addition to the COVID-19 pandemic, we met and are working to overcome several technological challenges that are out lined in the "Novel Avenues" section. Briefly, primer design and in-silico PCR analyses are challenging and time consuming, thus we initiated development of an Automated Primer Design Pipeline to reduce this burden allowing more robust assays to be generated and characterized with reduced hands-on time. Second, amplicon sequencing is inherently targeted allowing for increased sensitivity when compared to untargeted and metagenomic techniques, however, this requirement can be a challenge when responding to outbreaks of unknown etiology, thus, we conducted initial validation with incorporation of nontargeted bacterial assays into the Food-Path assay to supplement our targeted amplicon sequencing with untargeted 16S sequencing. Finally, another challenge to high throughput amplicon applications is verifying low level (i.e., low target burden) positives since these methods are highly sensitive to aberrations (e.g., low level contamination, index hoping, etc.), which can produce false positives and require artificially high cutoff values. To further control for this challenge, we designed and initiated testing the addition of unique synthetic DNA spike-ins (SDSI) into each sample for this and similar multiplexed amplicon sequencing assays. This process modification is showing us several advantages over our previous workflows, allowing for low level contamination to be detected and quantified while providing an internal control to low burden samples. Novel Avenues A. Primer Design Pipeline A time-consuming component of this project was creating and conducting in-silico analyses of primers. This highlighted a need for a streamlined process to more efficiently build and validate highly multiplexed amplicon sequencing assays. To accomplish this, we initiated development of a novel bioinformatic pipeline that will allow for more robust assays and tests to be built in less time using our automated primer design workflow. Additionally, we will be able to further explore high diversity targets (e.g.,Bacillus cereus) using a Kmer primer design workflow that will assess all regions within a genome for primer design. Although this work was not fully funded under this project and is currently partially complete, we have addressed the primer design challenges that we have encountered within this project and are incorporating solutions within this workflow (Figure 3). B. Incorporation of 16S assay Although amplicon sequencing has massive advantages to metagenomic and 16S sequencing techniques, such as increased specificity allowing higher sensitivity, cheaper processing costs, and faster data analysis, amplicon sequencing is only able to identify and characterize targets that are covered by the primer sets within the assay, an inevitable challenge. However, we have explored the addition of a 16S primer set into our Food-Path assay which would allow for a percentage (10-30%) of the reads to not be specific to the designed and targeted amplicons and allow for general bacterial identification, alleviating this challenge. Initial testing of the 16S primers was performed with sample isolates which yielded more uniform results than what would be expected in complex samples. Within these samples, results from 16S had 100% agreement with the Food-Path assay targets and the original specimen (Figure 4). We anticipate that this addition will improve Food-Path's capabilities especially when responding to food pathogen outbreaks that have multiple possible bacterial pathogen origins. C. Synthetic DNA Spike-Ins (SDSI) Finally, one challenge in high throughput amplicon applications is verifying low-level positives since these methods are highly sensitive to aberrations (i.e., low-level contamination, index hopping, etc.) that can produce false positives. In an attempt to lower our thresholds and increase data quality, we have designed a new set of synthetic controls that can be used to detect low-level sample-to-sample contamination. To create a flexible approach, we created a bioinformatic workflow that can generate nucleotide sequences which are then blasted across GenBank to ensure that they do not have similar sequences occurring within nature. These novel and randomly generated sequence fragments were then concatenated and separated by several locations, which could be used as reverse primers allowing for a variable-size fragment to be created and tuned to the specific sequencing workflow (Figure 5). We have tested this technology in several applications and believe that it provides a novel and flexible workflow that can be implemented in clinical and surveillance scenarios, where data quality is of utmost importance, while not drastically affecting the quality of the assay. This approach was tested opportunistically (with other funds) in WGS SARS-CoV-2 samples and yielded performance comparable to the Broad method and performance similar to samples where no spike-in was used (Figure 6). In addition to the opportunistic validation within SARS-CoV-2, we have begun to implement this into the Food-Path assay. To date, we have been titrating our concentrations of SDSI primer and SDSI template to yield a target of 5-10% of the total reads. We have had high success with this method; however, we are still exploring the needed concentration to yield consistent results in an amplicon setting. What opportunities for training and professional development has the project provided?Our Food-Path project utilizes targeted sequencing for precision genomic interrogation and analyses to address a critical public health issue. As these factors are ubiquitous in our mission and are continuously evolving, we've taken advantage of this opportunity to increase our talent capacity and build a more robust knowledge base and collective skill set. We have several research associates that are being trained in targeted sequencing methods and data analysis, as well as learning about foodborne pathogens, their toxins, and the most important mechanisms of AMR. Additionally, we've worked with our collaborators at Northern Arizona University and our contacts at Arizona on Montana Veterinary Diagnostic Laboratories for cross-education purposes. How have the results been disseminated to communities of interest?For this project we obtained environmental and animal samples from various collaborators and partners, including from farm and ranch owners and Veterinary Diagnostic Laboratories specializing in livestock. We have reported the results of our findings for pathogen species identification, AMR gene carriage, and even the relatedness between their samples (indicating transmission of a pathogen from one site to another) back to our project partners. Additionally, we consistently participate in outreach activities, including presentations and workshops to the general public, social media posts, programs aimed at high school, undergraduate, and graduate students, and Arizona's STEM programs. Lastly, we strive to share findings with the scientific community through publications. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Across the project period, we have completed assay design and validation for Food-Path including primer design,in-silicovalidation, wet lab testing of individual assays, multiplex optimization, sensitivity/specificity analyses and complex sample evaluation. On the bioinformatic side of the project, we developed and tested a Food-Path analysis module for our Amplicon Sequencing Analysis Pipeline (ASAP) bioinformatic tool. Additionally, we have explored and are continuing to explore additional novel avenues for amplicon sequencing. Assay Design and Validation A. Primer Design For this project, a total of 274 separate primers sets were designed or assessed for inclusion within the Food-Path assay. Overall, these assays detect and/or characterize ~100 different public health relevant food-borne pathogens, resistance mechanisms, and toxins. These assays cover the majority of targets within our original proposal with a few exceptions (e.i.,Bacillus cereusandClostridium botulinum). B. Wet Lab Single-plex Testing Prior toin-silicoassay validation, candidate assays underwent wet-lab testing in single-plex. These tests utilized real-time SYBR green PCR which allowed for confirmation of target amplification, detection of non-target amplification, and ranking of assay efficiency if there were multiple candidate assays available (Table 1). These data were used along with the in-silico validation to create a final multiplex primer set. C. In-silicoValidation In-silicoassay validation focused on utilizing TGen'sin-silicoPCR program to identify primer sets that had high inclusivity metrics (amplifying target organisms) while having high exclusivity metrics (not amplifying off target organisms). These data were then used to prioritize candidate assays for consideration within the final multiplex assay. Included assays for the Food-Path assays are included in Table 2. The majority of the assays had high inclusivity and specificity metrics, although the designed Astrovirus assay had an extremely low inclusivity metric and should be further evaluated through an exhaustive primer design pipeline to reduce the false negative rate (SeePrimer Design Pipeline). D. Multiplex Optimization Multiplex assay interactions were limited via the use of PrimerPooler which allowed for optimization of 2 primer-pools to minimize primer interactions (Table 1). E. Multiplex Validation Multiplex validation of the Food-Path assay focused on identifying the preliminary limit of detection of assays, identifying non-specific amplification, and understanding assay efficiency when multiple targets were available. Preliminary limit of detection (LoD) testing was completed by testing a serial dilution of positive controls in triplicate and identifying the dilution at which the assay lost sensitivity to the target and the target became undetectable. Non-specific interactions were identified by analyzing results for assays that were not expected to amplify within control samples. Characterization of the non-specific amplification allows for specific thresholds on breadth of coverage to be adjusted within our analysis process (ASAP) that limits the likelihood of false-positive detection. F. Off Target Amplification Analysis Off target amplification was tested by running the multiplex across a variety of organisms. No off-target amplification was consistently detected; however, a few samples did have evidence of low level off target contamination. The sensitivity of this technique requires inclusion of additional controls to detect and quantify this low-level contamination (SeeSynthetic DNA Spike-Ins). G. Preliminary Limit of Detection Testing Preliminary LoDs were tested by utilizing quantified concentrations of target organisms and identifying the dilution at which targets had a >5% false negative rate across 1-4 replicates (Table 3). Results are compiled below and show that the most of the assays have a low LoD (high sensitivity), however a few assays (e.g., Astrovirus and Cryptosporidium) were unsuccessful in detection of the target organisms and will be further developed. H. Complex Sample Evaluation Complex sample evaluation was conducted using cattle fecal samples collected from a farm, milk samples and several meat samples. The final Food-Path assay was run across all of these samples including samples extracted with numerous replicates (Figure 1). Overall,Staphylococcus aureuswas the most common target detected in both fecal and milk collections. Additionally, the Food-Path assay detected numerous samples withmecAresistance mechanism which is located on a commonS. aureusplasmid. Within the fecal samples in addition toS. aureus,Giardia,E. coli, and Campylobacter jejuniwere detected. I. Utilization in Outbreak Setting In addition to the testing above, the Food-Path assay was used to detect possible Cyclospora contamination in leafy green material that was suspected in a recent Arizona Cyclospora outbreak under investigation by AZ Department of Health Services. Analysis also included remnants of a Greek salad from a Cyclospora case. The Food-Path Cyclospora assay was negative for the leafy greens but produced limited signal on PCR, and less than 20 amplicon reads for the suspect salad. The negative leafy greens and indeterminant salad results were consistent with results from the published FDA Cyclospora PCR assay and metagenomic analysis on the same samples further supporting Food-Path's applicability in public health responses. Results from this analysis were shared with local and state health and the CDC. Bioinformatic Improvements Development to Amplicon Sequencing Analysis Pipeline (ASAP) In addition to assay design and validation, we have completed significant changes to our software tool for amplicon sequence analysis, Amplicon Sequencing Analysis Pipeline (ASAP). We have incorporated the ability to set identity thresholds on a per-assay basis, which will add accuracy in counting the reads that align to our reference sequences. These thresholds will distinguish conserved DNA sequence across species that is not necessarily specific to our target, e.g., thegyrA gene in which mutations can confer AMR but that is found in multiple species. We have also built in the capability to assess results from multiple assays to characterize a single sample feature, by combining the assay using AND/OR/NOT operators. For example, if a sample is positive on theE. coliassay, the strain type ST131 assay, theblaCTX-M assay, and for a quinolone resistance-conferringgyrA mutation, we can be fairly confident the sample contains the widespread and virulent H30-Rx subclone ofE. coli. Additionally, we have created a custom HTML report format (Figure 2), as well as an R-script for parsing the ASAP output and generating heat maps of the results (Figures 1).
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Presentation Title: "Fully automated k-mer based primer design for highly multiplexed, pan-taxonomic group assays."
Conference/Meeting: 17th Annual Sequencing, Finishing, & Analysis in the Future Conference (Santa Fe, NM)
Date: June 6th, 2023
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Presentation Title: ASAP: A customizable Amplicon Sequencing Analysis Pipeline for high-throughput characterization of complex samples
Conference/Meeting: CDC Advanced Molecular Detection-TOAST (virtual)
Date: November 4th, 2022
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Progress 04/15/21 to 04/14/22
Outputs
Target Audience:This Food-Path surveillance project is designed to detect potential human and animal health threats in the entirety of our food production system (from farm to table). As such, our target audience is broad. Our main goal is to develop a tool for public health and food/water safety agencies to use proactively in the continuous monitoring of the food system for potential health threats. Thus, the target beneficiaries are anyone that consumes food. Changes/Problems:The most significant issue we've experienced with this project is our significant pivot to respond to the COVID-19 pandemic. As with other laboratories, institutes, and agencies, the pandemic assumed much of our time, efforts, and resources. Though we have been successful in our pandemic response, we look forward to restoring our momentum on this and other important public health projects. For this reason, we have applied for a 1-year no-cost extension in order to meet the objectives originally outlined in our proposal for this project. What opportunities for training and professional development has the project provided?Our Food-Path project utilizes targeted sequencing for precise genomic interrogation and analyses to address a critical public health issue. As these factors are ubiquitous in our mission and are continuously evolving, we've taken advantage of this opportunity to increase our talent capacity and build a more robust knowledge base and collective skill set. We have several research associates that are being trained in targeted sequencing methods and data analysis, as well as learning about foodborne pathogens, their toxins, and the most important mechanisms of AMR. Additionally, we've worked with our collaborators at Northern Arizona University and our contacts at the Veterinary Laboratories for cross-education purposes. Most importantly, a core overarching value at TGen North is fostering an environment of ongoing professional growth. How have the results been disseminated to communities of interest?For this project we are obtaining environmental and animal samples from various collaborators and partners, including from farm and ranch owners and a Veterinary Laboratory specializing in livestock. We intend to report back to these partners the results of our findings for pathogen species identification, AMR gene carriage, and even the relatedness between their samples (indicating transmission of a pathogen from one site to another). Additionally, we at TGen North consistently participate in outreach activities, including presentations and workshops to the general public, social media posts, programs aimed at high school, undergraduate, and graduate students, and Arizona's STEM programs. Lastly, we strive to share findings with the scientific community through publications. What do you plan to do during the next reporting period to accomplish the goals?The first quarter of the 2022-2023 year we will complete the initial Food-Path assay panel validation, including multiplex evaluation, and make any necessary adjustments in the second quarter. Also in the first half of the year we will add to our isolate repository and begin whole genome sequencing, in order to conduct phylogenetic analysis of particular food-borne pathogens in the second half of the year. Also in the second half of the year, we will finish the Food-Path assay and ASAP software evaluation on samples collected from different points of the food system, including those collected from a ranch and retail food. Below is our proposed task timeline for the 1-year no-cost extension period from 2022-2023. 2022-2023 Tasks for Objectives 1-3 Q1 Q2 Q3 Q4 1 Assay design and in silico validation for B. cereus and any failed assaysHeavy Medium 1-2 in vitro validation of assays with pure genomic DNA, including vet isolatesHeavy Medium 2 Upstream assay evaluation with ranch samples Medium Heavy Medium 2 Downstream assay evaluation with retail food samples Medium Heavy Medium 3 Building isolate repository for whole genome sequencing Heavy Heavy Medium Medium 3 Whole genome sequencing of study isolates Heavy Heavy Medium 3 Phylogenetics of isolates within population of genomes in public databases Medium Heavy 3 Culture of select complex samples for mixture validation of ASAP Heavy Heavy
Impacts
What was accomplished under these goals?
In 2021-2022, we completed the primer design and in silico validation for the amplicon sequencing assay panel, including primers targeting all of the food-borne pathogens listed in our original proposal - with one exception for Bacillus cereus. (This primer design has been a challenge due to the complexity of its taxon.) This set will be completed in 2022-2023. We have multiple primer sets that passed in silico validation for each pathogen and AMR gene being targeted, to 1. account for dropouts during laboratory validation of sensitivity and specificity, and 2. build in redundancy to ensure sensitivity when screening complex or low-titer samples. We are currently querying our isolate collection to identify known positive controls for the AMR target primer sets. (Positive control isolates have already been identified for the species identification primer sets.) And we have begun initial laboratory validation of the primer sets, in single- and in multiplex, on known positive samples. Additionally, we have started laboratory validation on a published quality control methodology that will identify any cross-contamination between samples in a set being processed and sequenced at the same time. As the application of the Food-Path assay is intended to be high-throughput, this may be an important quality control measure to ensure accurate results. The methodology uses Synthetic DNA Spike-Ins (SDSIs, https://www.nature.com/articles/s41564-021-01019-2), and is a measure we are considering incorporating into all of our multiplexed amplicon sequencing assays, especially those for translation into clinical use. Lastly, we've made significant progress on the software tool for amplicon sequence analysis, ASAP. We have incorporated informatics to set thresholds, by default or user-determined, that will add accuracy in counting the reads that align to our reference sequences. These thresholds will distinguish conserved DNA sequence across species that is not necessarily specific to our target, e.g., the gyrA gene in which mutations can confer AMR but that is found in multiple species. We have also built in the capability to assess results from multiple assays to characterize a single sample feature. For example, if a sample is positive on the E. coli assay, the strain type ST131 assay, the blaCTX-M assay, and for a quinolone resistance-conferring gyrA mutation, we can be fairly confident the sample contains the widespread and virulent H30-Rx subclone of E. coli.
Publications
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Progress 04/15/20 to 04/14/21
Outputs
Target Audience: This Food-Path surveillance project is designed to detect potential human and animal health threatsin the entirety of our food production system (from farm to table). As such, our target audience isbroad. Our main goal is to develop a tool for public health and food/water safety agencies to useproactively in the continuous monitoring of the food system for potentialhealth threats. Thus, the target beneficiaries are anyone that consumes food. Changes/Problems:The most significant issue we've experienced with this project is our significant pivot to respond to the COVID-19pandemic. As with other laboratories, institutes, and agencies, the pandemic assumed much of our time, efforts, and resources. Though we have been successful in our pandemic response, we lookforward to restoring our momentum on this and other important public health projects. What opportunities for training and professional development has the project provided?Our Food-Path project utilizes targeted sequencing for precise genomic interrogation and analyses to address a critical public health issue. As these factors are ubiquitous in our mission and are continuously evolving, we've taken advantage of this opportunity to increase our talent capacityand build a more robust knowledge base and collective skill set. We have several researchassociates that are being trained in targeted sequencing methods and data analysis, as well aslearning about food-borne pathogens, their toxins, and the most important mechanisms of AMR.Additionally, we've worked with our collaborators at Northern Arizona University and our contactsat the Veterinary Laboratories for cross-education purposes. Most importantly, a core overarchingvalue at TGen North is fostering an environment of ongoing professional growth. How have the results been disseminated to communities of interest?For this project we are obtaining environmental and animal samples from various collaborators andpartners, including from farm and ranch owners and a Veterinary Laboratory specializing inlivestock. In previous years we've been able to report back to these partners the results of ourfindings for pathogen species identification, AMR gene carriage, and even the relatedness betweentheir samples (indicating transmission of a pathogen from one site to another). Due to pandemicresponse work, we did not produce these kind of data. However, we are resuming our work in thisarea for the 2021-2022 grant year. Additionally, we at TGen North consistently participate in outreach activities, includingpresentations and workshops to the general public, programs aimed at high school, undergraduate, and graduate students, and Arizona'sSTEM programs. What do you plan to do during the next reporting period to accomplish the goals? For the 2021-2022 grant year, we are already finalizing the assay development for the Food-Pathamplicon sequencing assay panel and the customization of our data analysis tool, ASAP, for rapidturnaround of sample results. Finally, we arelatest knowledge-base of the target genes and possible orthologs andl mutations, and we have a minimal number of targets left to pursue (i.e., Bacillus cereus).
Impacts
What was accomplished under these goals?
As an institute committed to pathogen research in the public health arena, much of 2020 was focused on the COVID-19 pandemic response. Although direct progress on our Food-Path system was inhibited in 2020, with our success in the pandemic response we indirectly made progress on all of our translational projects, including this one. For the COVID-19response work, TGen North set up a CLIA-registered and FDA-certified laboratory to test patient samples for diagnosis. With this experience, we have now used the protocols and systems in place to advance our other translational prototypes, moving these projects a step closer to validation and widespread use. Although much of 2020 and early 2021 were focused on emergency response to the pandemic, we did make progress on this project. Whereas in previous years our focus was on the acquisition of isolates and specimens to test and validate the Food-Path system, in 2020-2021 we worked on the development of additional assays to add to our Food-Path multiplexed panel, including adding several pathogens (e.g., Bacillus cereus, Clostridium difficile, Listeria monocytogenes), a genomic serotyping system for Salmonella, and AMR targets relevant to the new pathogen additions. Finally, we are continuallymonitoring the epidemiology of AMR and food-borne pathogens in the U.S.
Publications
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Progress 04/15/19 to 04/14/20
Outputs
Target Audience:Public health officials: We have met with state public health officials to describe the project and to let them know that we will provide any relevant public health related data (e.g., information of detected outbreaks, etc) that may arise during our analyses. Ranchers: Crystal Hepp (NAU PI) has received samples from the Hepp family ranch and has thoroughly described the project with pertinent ranch staff. We will provide information to them on any results from the samples collected from their ranch. Veterinary Officials: We have communicated with the Montana and Arizona State Veterinary Diagnostic Labs as to the purpose of the project and have worked with them to identify useful samples to provide and associated metadata. At this point, we have limited results of interest to communicate to our partners. However, we have informed them that the assays we have developed do work on the positive samples they provided. We are planning to disseminate our early findings that the Annual Southwest One Health Symposium (planned for December 2020) and will disseminate final findings though publication in peer-reviewed journals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have trained three undergraduates and one graduate student during the second year of funding. The undergraduates have gained culturing, polymerase chain reaction (PCR), and quantitative PCR (qPCR), amplicon library preparation and sequencing skills. The graduate student has learned how to perform 16S microbiome analysis. One undergraduate was invited to present at the National Conferences on Undergraduate Research, which was unfortunately cancelled due to the COVID-19 pandemic. How have the results been disseminated to communities of interest?Public health officials: We have met with state public health officials to describe the project and to let them know that we will provide any relevant public health related data (e.g., information of detected outbreaks, etc) that may arise during our analyses. Ranchers: Crystal Hepp (NAU PI) has received samples from the Hepp family ranch and has thoroughly described the project with pertinent ranch staff. We will provide information to them on any results from the samples collected from their ranch. Veterinary Officials: We have communicated with the Montana and Arizona State Veterinary Diagnostic Labs as to the purpose of the project and have worked with them to identify useful samples to provide and associated metadata. At this point, we have limited results of interest to communicate to our partners. However, we have informed them that the assays we have developed do work on the positive samples they provided. We are planning to disseminate our early findings that the Annual Southwest One Health Symposium (planned for December 2020) and will disseminate final findings though publication in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?We will attempt to multiplex the 128 assays, as well as additional assays, that we developed in year 2. We will culture and whole genome sequence target organisms from all positive ranch, store-bought samples, and Veterinary Diagnostic Laboratory samples. We will collect additional samples from the Hepp Ranch (original samples collected in year 1) to determine if the same strains of target pathogens are present year after year. We will collect additional samples from commercial and farmers markets and use the Food-Path Assay to identify target pathogens in community circulation.The Food-Path system will result in a unique invention, that will be available for licensing for manufacture and distribution. Individual assays will each provide opportunity for additional marketable products.
Impacts
What was accomplished under these goals?
Although we experienced the universal slowdown of scientific research due to the COVID-19 pandemic, we did have significant impact on the Y2 goals: We have secured diagnostically positive samples, from local ranching and farming operations, for our target pathogens from the Montana Veterinary Diagnostic Laboratory. We attempted to obtain similar samples from the AZ Veterinary Diagnostic Laboratory, however, their access to such samples is severely limited - they will however inform of any relevant samples they receive. We have determined which of our target pathogens are present in manure, soil, water, cattle fecal and vaginal swabs, and cattle milk samples using 16S, ITS, and Food-Path sequencing. This is informing our culturing methods to move towards whole genome sequencing of Salmonella, Campylobacter, E. coli, Listeria monocytogenes and Clostridium discussed in our 3rd aim. Using our in-house bacterial quantification assay, we've determined that bacteria in raw meat is best identified by swabbing the meat followed by extraction using Qiagen's DNeasy Blood and Tissue kit and including an overnight lysis. We have developed 128 assays to identify target pathogens and are testing them on positive controls as well as field collected samples that are positive for the target organisms. We have updated the ASAP bioinformatics tool to include all targets in the Food-Path Assay, including: E. coli 01, Shigella, Enterococcus faecalis, E. faecium, toxin assays for stx1,stx2, and eaeA virulence genes, Salmonella enteritica and serotype specific targets, Clostridium and Vibrio species, Cronobacter spp., Norovirus, Astrovirus, and Giardia, Cyclospora and Cryptosporidium.
Publications
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Progress 04/15/18 to 04/14/19
Outputs
Target Audience:Public health officials: We have met with state public health officials to describe the project and to let them know that we will provide any relevant public health realted data (e.g., information of detected outbreaks, etc) that may arise during our analyses. Ranchers: Crystal Hepp (NAU PI) has received samples from the Hepp family ranch and has througroughly decribed the project with pertninet ranch staff. We will provide information to them on any results from the samples collecte from their ranch. Veterinary Officials: We have communicated with the Montana State Veterinary Diagnostic Lab as the the purposoe of the project and have worked with them to identiy useful samples to provide and associated metadata (e.g., bacterial MICs) Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have trained three undergraduate interns in the process of amplicon library preparation and sequencing. How have the results been disseminated to communities of interest?As described under target audiences, we will provide updates to the project parnters including ranch and MVDL officials, as well as to public health partners. We anticiapte initial resultes being available in Year 2. What do you plan to do during the next reporting period to accomplish the goals?1. We plan to complete the remianing assay development, including assays for Clostridium and Vibrio species, Norovirus typing, Astrovirus, and the parasites (giradia, Cyclospora and Cryptosporidium). 2. We plan to update the ASAP bioinformatic toolfor amplicon sequence analysis to include all targets in the Food-Path Assay 3. We plan to initiate testing of preliminary samples from year 1. 4. We plan to continue to optimaize our complex samples culture, enrichment and extraction methodologies. 5. We plan to secure the well characterized isoaltes from the Montana Veterinary Diagnostic Lab that we have disucssed with them during Year 1.
Impacts
What was accomplished under these goals?
1. We designed assays for E. coli 0157, Shigella, Enterococcus faecalis, E. faecium.We have designed toxin assays for stx1, stx2, and eaeA virulence genes. We have initiated design for the Salmonella enteritica serotypes. 2. In order to complete our assay validation panel, we have obtained multiple new isoaltes from theATCC and BEI repositories, including: Enterococcus hirae, Cronobacter spp., Norovirus, Astrovirus, and Cycolospora cayetanensis.We have made arrangements for a shipment of well characterized isoaltes from the Montana State VDL in Year 2. 3. In order to test Food-Path ability to detect target organisms in complex samples, we have obtained manure, soil waterand cattle swab samples from the Hepp Ranch in Montana, andfood meat from multiple llocal grocery stores, including, ground beef, chicken breasts, and pork chops. We are currently testing multiple culture methods, including an improvised stomacher bag culture system,to enrich for any present bacteria. Primary samples, enriched culture material and isolates will be screened with the Food-Path assay in Year 2. Isolates will also be sequenced for phylogenetic analysis in Year 3
Publications
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