PARTNERSHIP: DEVELOPMENT OF A NANOPARTICLE-ENABLED ENRICHMENT AND DNA-BIOSENSING FOR ONSITE MONITORING OF MULTIPLE FOODBORNE PATHOGENS IN LARGE SAMPLES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1027815
• Grant No.: 2022-67017-36982
• Cumulative Award Amt.: $769,000.00
• Proposal No.: 2021-08313
• Project Start Date: May 15, 2022
• Project End Date: May 14, 2025
• Grant Year: 2022
• Program Code: [A1332]- Food Safety and Defense

Project Director
Alocilja, E.

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
BIOSYSTEMS AG EGR

Non Technical Summary
According to a USDA Economic Research Service report in 2018, the economic burden due to 15 foodborne pathogens was estimated close to $18 billion in the U.S. Salmonella and Campylobacter caused the most reported bacterial foodborne illnesses. Epidemiological studies have suggested that products of poultry origin are among the most common vehicles for transmission of Salmonella spp. and Campylobacter spp.. In the U.S., poultry meat consumption is increasing per capita per year compared to the rest of the world. Bacteria from farm production, slaughterhouses, and processing equipment contaminate carcasses and processed meat products. The presence of very low levels of live bacteria in food samples can render the food hazardous. Microbiological culture-based methods are limited in their ability to provide timely data. Currently available rapid detection systems are often laborious, expensive, and require laboratory facilities. They also lack the ability to quickly enrich and detect targets in large samples. There is an unmet need for rapid, low-cost, onsite, and accurate detection of foodborne agents in large samples. Thus, the goal of this proposal is to rapidly monitor the temporal dynamics of Salmonella and Campylobacter infection in poultry farms and slaughter facilities using a nanoparticle-based biosensor with capabilities for rapid and simultaneous enrichment, detection, and reporting of multiple agents.This research is a partnership between Michigan State University and Tuskegee University for food safety. Due to its simplicity and affordability, the cellphone-enabled SMART biosensor can be used onsite to monitor and generate internal information on the effectiveness of antimicrobial intervention strategies, provide temporal dynamics of the infection rate at control points, and provide early information on potential development of antimicrobial resistance of the bacteria to the treatment and prevention strategies.Although the biosensor may not be able to replace culture and may not be as sensitive as real-time polymerase chain reaction (RT-PCR), it will allow high frequency testing and support faster decision-making by producers and operators, resulting in the implementation of pathogen reduction programs that will prevent these pathogens from entering the food supply chain, thus protecting public health, reducing foodborne illness outbreaks, and sustaining the availability of nutritious foods. The biosensing technology will also be able to guide producers identify specific sites in their facilities that need improved sanitation and intervention strategies, resulting in higher efficiency of operation, increased productivity, and overall higher business profitability.

Animal Health Component

40%

Research Effort Categories

Basic

20%

Applied

40%

Developmental

40%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3299	1040	30%
723	3299	1100	30%
404	3299	2020	40%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 404 - Instrumentation and Control Systems; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1040 - Molecular biology; 2020 - Engineering; 1100 - Bacteriology;

Keywords

food safety

biosensor

chicken meat

extraction

poultry

Goals / Objectives
Poultry is one of the most consumed animal-based protein in the U.S. Unfortunately, poultry and poultry products are considered as major vehicles of Salmonella and Campylobacter infection in humans. Salmonella and Campylobacter are the top two leading causes of bacterial foodborne diarrheal illnesses in the U.S. causing close to a million cases of illnesses by each organism every year. According to a USDA Economic Research Service report in 2018, the economic burden was $4 billion due to Salmonella (non-typhoidal) infection and $2 billion due to Campylobacter infection. Currently, there is a technology gap for the simultaneous extraction, concentration, purification, and detection of multiple pathogens in field settings, close to the contamination site. Thus, the goal of this proposal is to rapidly monitor the temporal dynamics of Salmonella and Campylobacter infection in poultry farms and slaughter facilities using a nanoparticle-based biosensor with capabilities for rapid and simultaneous enrichment, detection, and reporting of multiple agents. The specific objectives are: (1) optimize the Site-enriched-Multi-Array-Reporting Biosensing Technology (SMART) for rapid detection of Salmonella spp. and Campylobacter spp. in large samples; (2) optimize a cellphone-based data capture, analysis, and reporting of the biosensor signal output; and (3) validate the cellphone-enabled SMART biosensor for simultaneous detection of Salmonella spp. and Campylobacter spp. in selected poultry farms and slaughter facilities.

Project Methods
The specific objectives of this project are: (1) optimize the Site-enriched-Multi-Array-Reporting Biosensing Technology (SMART) for rapid detection of Salmonella spp. and Campylobacter spp. in large samples; (2) optimize a cellphone-based data capture, analysis, and reporting of the biosensor signal output; and (3) validate the cellphone-enabled SMART biosensor for simultaneous detection of Salmonella spp. and Campylobacter spp. in selected poultry farms and slaughter facilities.The SMART biosensor combines microbial enrichment, biosensing assay, and smartphone-based data analytics designed to empower proactive and effective decision making in food safety. The microbial enrichment step is facilitated by the glycan-coated magnetic nanoparticles (GMNPs); the diagnostic test is facilitated by the thiolated gold nanoparticles (GNPs) for the detection of multiple target agents and genes. GMNPs are used to enrich the microbial contaminants in the sample; GNPs are used to detect and confirm the presence of multiple pathogens and multiple target genes in the sample. The solution changes color after adding a signal buffer: red for the presence of the target DNA due to the formation of bacteria-specific complex; blue if the target DNA is absent. The biosensor can be conducted at higher frequency due to its simplicity, speed, and affordability. Although GMNPs lack the specificity to the pathogens, they have shown the capacity to extract and concentrate multiple bacteria, increasing the sensitivity of the DNA-based biosensing assay.Target genes for Salmonella spp. and Campylobacter spp. will be identified. The invA gene has been frequently used in biomolecular detection of Salmonella spp.. Additional genes will be explored, such as hilA, fljB, fliC, bcfD, phoP, siiA, and others as necessary. Specific Campylobacter genes, such as omp50, mapA, ceuE, and flaA, could be used for specific detection of C.jejuni and C. coli. Briefly, sequences of these genes will be retrieved from the NCBI microbial genome-sequencing database. Unique targets will be identified after thorough study of multiple alignment of these sequences. Once target sequences are identified, a BLAST search directly from NCBI and PATRIC databases will be performed to verify the uniqueness of the target sequence. The target DNA will further be validated for their specificity. We will use multiple target genes and multiple oligonucleotide probes of various lengths in the SMART biosensor.For objective 1, the following studies will be conducted to optimize the enrichment and biosensing assays under lab conditions:Optimize sampling protocols for collecting manure samples from poultry farms and scald-water samples from slaughterhouses.Optimize sample preparation that is compatible with enrichment and biosensing assaysConduct multi-Array extraction and enrichment within 10-20 minDetermine the concentration factor of the enrichment assayConduct genomic DNA extractionConduct multi-array DNA detection within 30-40 minDetermine the analytical sensitivity of the biosensorDetermine the analytical specificity of the biosensorDetermine the analytical precision of the biosensorDetermine the analytical accuracy of the biosensorValidate the biosensor performance using PCRFor objective 2, the following studies will be conducted to optimize the smartphone-based data capture and analysis:Design and code the algorithm for data capture using the smartphone cameraDesign and code machine learning algorithm for data analysisDesign and code the App for analytical output displayDesign and code the App for user-friendly operation and human-machine interfaceDesign and code the App for display of temporal dynamics of infectionFor objective 3, the following studies will be conducted to assess the performance of the enrichment and biosensing assays in farm and field samples:Optimize sample collection from farm and slaughterhouse facilitiesCollect fecal and water samples from various locations in farms and slaughterhousesConduct multi-array enrichment on-farmConduct genomic DNA extraction on-farmConduct multi-array DNA detection on-farmCompare biosensor results with PCRMonitor the temporal dynamics of infection due to Salmonella and CampylobacterActivities cutting across the three objectives -System development to assess technology readiness and adoption by the poultry industry:Assess compatibility of enrichment and biosensing assays with industry practice to ensure smooth adoption of technologies into the farm and slaughterhouse operationMeet with collaborators and other stakeholders to assess technology readinessAssess the benefits, costs, competitors, constraints by potential users, and other parameters to assess the readiness of the technology for on-farm useBenchmark other existing tests, such as culture and PCR, with biosensor technologyMeet with representatives of industry associations, poultry extension specialists, and other stakeholders to evaluate the significance of the findings, and how that information will impact decision making at specific sites in the field

Progress 05/15/23 to 05/14/24

Outputs
Target Audience:Industry collaborators: During the reporting period, we worked with our industry collaborators, the Michigan Turkey Producers and Miller Poultry. We collected samples from their processing plants, the Doherty Turkey Farm, and a Miller Poultry farm contractor in Indiana. We also contacted turkey and poultry farms in Alabama to validate the testing of Salmonella serovar. General public: We presented our research results to the public at the MSU Science Festival in April 2024, which schoolchildren from K-12 and parents from around the state of Michigan attended. We interacted with the rural farmer community in West Alabama. Students: We trained undergraduate, DVM, and graduate students and visiting scholars during the reporting period. Professional societies: We presented our results at the following conferences: Institute of Biological Engineers (IBE) in Iowa American Society of Agricultural and Biological Engineers (ASABE) in Nebraska International Association for Food Protection (IAFP) in Canada Phi Zeta Research Symposium in Alabama Multistate committee NC-1194 (Nanotechnology and Biosensors) in Florida Comments from our stakeholders, 2023-2024: "We continue to be very excited about the rapid detection of Salmonella with the technology you are developing for our industry. We believe the technology will be very useful when monitoring the cleanliness of finishing barns after clean-out, between flocks. It is very important to our growers to have our barns as clean as possible when moving flocks from the brooders to the finishing barns, the use of the rapid technology can help the farm managers assess the cleanliness of the barns prior to movement. The 4-hour result time allows our farms to have time to re-clean and re-test areas of concern, allowing our birds the healthiest conditions possible. We are considering using the technology as a verification tool for our load-out crews. While we know that Salmonella resides in the gut of our birds, we also believe they will not shed the Salmonella unless we create an environment that causes them stress. If a barn tests negative prior to load-out, and positive after load-out, the data can be used for continuous improvement to better understand the things that cause our birds stress." Tina Conklin, Michigan Turkey "Continuous improvement in food safety in the area of pathogen control is of utmost importance. Timeliness, accuracy, and valid measurements of our processes are critical for quick and accurate assessments and decision making. Pathogens are grown and introduced to live poultry at the farm. It is beneficial to have quick and accurate measurement techniques on the farm to allow for faster intervention to greatly reduce the likelihood and the level of pathogens from reaching final product destined for consumers." Roger Stearns, Miller Poultry Changes/Problems:Acquiring and maintaining Campylobacter cultures have been challenging due to the specific atmosphere requirement and the preparation of the special media, which requires different cocktails of supplements. These challenges made the in vitro assays difficult. After experiencing these challenges, we have since adjusted our approach and considered the organism's special growing conditions. What opportunities for training and professional development has the project provided?Training opportunities on this project were provided to: Nine graduate students (3 MSU, 6 TU) Seven undergraduate research assistants (4 during the semesters, 3 in summer, all at MSU) One visiting scholar (1 MSU) Two professional DVM students (2 TU) Two of our students received awards, demonstrating the high quality of students in this project. Anthony James Franco, 2023. Best Poster Award, 2023 Engineering Graduate Research Symposium, Michigan State University, East Lansing, MI 48824, Aug. 22, 2023 Anthony James Franco, 2023. First Place winner, Data and Research Translation Award, 2023 Engineering Graduate Research Symposium, Michigan State University, East Lansing, MI 48824, Aug. 22, 2023 Devyn Hill, 2023. AGEP Conference, 2nd Place Oral Presentation for undergraduates, Michigan State University, East Lansing, MI 48824 How have the results been disseminated to communities of interest?Results from this project were disseminated through peer-reviewed journal publications and presentations at technical conferences. For journal publications, see the list of papers. For presentations, see the list below. Presentations at Professional Conferences: Franco AJ, Conklin T, Williams Z, Schweihofer J, Abebe W, and Alocilja EC, 2023. Rapid Salmonella Detection Using Nanoparticles: A Promising Technology for Safer Poultry Products, Poultry Summit, Atlanta, Georgia, Nov. 7-9, 2023 NC-1194 Committee meeting, Gainsville, Florida, Aug. 7-8, 2023 Franco AJ, Mayaka R, Abebe W, and Alocilja EC. 2023. Genomic Detection of Salmonella in Poultry Samples Using an End-to-End Nano-Biosensor Platform, International Association for Food Protection Annual Meeting 2023, Toronto, Canada, July 16-19, 2023 Caliskan-Aydogan, O., Sharief, S. A., & Alocilja, E. C. Nanoparticles for multiplex genomic detection of Carbapenem-Resistant E. coli in Foods, International Association for Food Protection (IAFP) International Meeting, Toronto, Ontario, Canada, July 16-19, 2023. Franco AJ, Conklin T, Schweihofer J, Williams Z, Alocilja EC. 2023. End-to-End Nano-Biosensor Platform for the Genomic Detection of Salmonella in Poultry Farm Samples, ASABE International Meeting, Omaha, NE, July 9-12, 2023 Mayaka R and Alocilja EC. 2023. A nano biosensor for the rapid detection of BlaNDM-1 Gene, ASABE International Meeting, Omaha, NE, July 9-12, 2023 Sharief S, Caliskan-Aydogan O, and Alocilja EC. 2023. PCR-less detection of E. coli from fresh produce using carbohydrate-coated nanoparticles, ASABE International Meeting, Omaha, NE, July 9-12, 2023 Caliskan-Aydogan O and Alocilja EC. 2023. DNA-Based Plasmonic Biosensor for KPC-producing Carbapenem-Resistant Bacteria, ASABE International Meeting, Omaha, NE, July 9-12, 2023 Alocilja, EC and Caliskan-Aydogan 2023. Nano-Biosensor for Genome-To-Phenome Profiling of AMR (G2PAR), Institute of Biological Engineers, Ames, Iowa, April 13-15, 2023 Franco AJ and Alocilja, EC 2023. Magnetically-Activated Cell Enrichment (MACE) for Improved Salmonella Detection, Institute of Biological Engineers, Ames, Iowa, April 13-15, 2023 Kierney Burks, Phi Zeta Research Symposium, College of Veterinary Medicine, Tuskegee University, September 2023 Presentations at MSU: Kao K, Cayen J, and Alocilja EC. 2023. Testing for Virulence Genes in E. coli O157 Using DNA Biosensor, University Undergraduate Research and Arts Forum (UURAF), Michigan State University, April 14, 2023 Heinecke K, Franco AJ, Alocilja EC, 2023. Magnetic Nanoparticle-Aided Rapid Screening of Microbial Contamination, University Undergraduate Research and Arts Forum (UURAF), Michigan State University, April 14, 2023 Zaborney Kline C and Alocilja EC, 2023. Microscopic Imaging of MNP-Bound Bacteria in Food Matrices, University Undergraduate Research and Arts Forum (UURAF), Michigan State University, April 14, 2023 Bieszke L, Franco AJ, and Alocilja EC. 2023. Extraction and Concentration of Salmonella from Poultry Plant Samples Using Magnetic Nanoparticles, University Undergraduate Research and Arts Forum (UURAF), Michigan State University, April 14, 2023 Wang P, Franco J, and Alocilja EC. 2023. Mid-SURE, Mid-SURE, Michigan State University East Lansing, MI. June 20, 2023 Amponsah E and Alocilja EC. 2023. Mid-SURE, Mid-SURE, Michigan State University East Lansing, MI. June 20, 2023 Hill D, Franco J, and Alocilja EC. 2023. Magnetic Nanoparticles (MNPs) for the enrichment of Salmonella enteritidis in turkey meat samples, Mid-SURE, Michigan State University East Lansing, MI. June 20, 2023 Caliskan-Aydogan, O., Sharief, S. A., & Alocilja, E. C. Carbohydrate-Coated Nanoparticles for PCR-less Detection of Salmonella from Chicken Meat. 2023 Engineering Graduate Research Symposium, Michigan State University, East Lansing, MI, May 11, 2023 Franco, A. J., Conklin, T, & Alocilja, E. 2023. Where Does Salmonella Come From? Preliminary Monitoring of the Presence of Salmonella at Various Stages of Poultry Processing. 2023 Engineering Graduate Research Symposium, Michigan State University East Lansing, MI, May 11, 2023. Recipient of Best Poster Award and First Place Data and Research Translation Award Hill D, Franco J, and Alocilja EC. 2023. AGEP Student Success Conference, Michigan State University, East Lansing, MI 48824, Nov. 9-11, 2023. 2nd Place Oral Presentation for undergraduates National Resources: Alocilja, E.C. 2023. Featured in Curious About Careers, Public Broadcasting Service (PBS) WKAR. Curious About Careers is a local public television program presented by WKAR. What do you plan to do during the next reporting period to accomplish the goals?Plans for next year include: Optimize the smartphone application for image display and analysis for the biosensing assay. Validate the magnetic enrichment in collaborators' farms & processing plants - extraction onsite. Validate the SMART biosensor in collaborators' farms & processing plants - biosensor tested onsite. Validate and compare the biosensor with Neogen's DNA kit. Generate data for temporal dynamics analysis. Calculate costs associated with the biosensor production and use in farms and processing plants. Calculate benefits associated with the biosensor technology compared with existing technologies. Assess the initial technology readiness of the biosensor. Meet with collaborators and other stakeholders to assess the significance of results and technology readiness.

Impacts
What was accomplished under these goals? Objective 1: Optimize the Site-Enriched Multi-Array Reporting Biosensing Technology (SMART) for rapid detection of Salmonella and Campylobacter in large samples. Proposed Activity: Confirm target genes for S. enterica serovars and Campylobacter jejuni and C. coli detection. Accomplishments: The invA gene was the target for Salmonella enterica; Cj0415 and cadF genes were the targets for Campylobacter jejuni. Various molecular regions of the genomes of multiple Salmonella serovars were investigated to identify other potential target genes. Objective 2: Optimize cellphone-based data capture, analysis, and reporting of the biosensor signal output. Proposed Activity: Optimize app or image/data capture, analysis, and display. Accomplishments: Improvements to the smartphone application user interface and image analysis algorithm were implemented and validated. A database for data integration from various locations and collaborations is being set up. Objective 3: Validate the cellphone-enabled SMART biosensor for simultaneous detection of Salmonella spp. and Campylobacter spp. in selected poultry farms and slaughter facilities. Proposed Activity: Validate the magnetic enrichment in samples sent to the laboratory. Accomplishments: Samples were collected from the turkey and poultry processing plants, a turkey farm, and a chicken farm. Magnetic enrichment studies were conducted on various field samples such as poultry rinsate, manure, turkey swabs, and ground turkey meat. The extraction and concentration factors of the magnetic nanoparticles were determined. Inoculation was performed in samples with no bacterial growth to determine the extraction and concentration factors of the magnetic nanoparticles. Improvements in the procedure, such as lower enrichment volume and longer incubation time, are currently being conducted. Proposed Activity: Optimize and validate the SMART biosensor in samples sent to the laboratory. Accomplishments: Biosensor assays were performed on fecal, manure, and carcass swab samples. Proposed Activity: Determine the sensitivity and specificity of the biosensor. Accomplishments: Sensitivity and specificity were determined using multiple fecal samples (raw and artificially inoculated) to identify Salmonella and other priority pathogens from a complex matrix. Selectivity and sensitivity assays were performed in vitro using previously designed probes. Validation in additional probes is currently being performed. Proposed Activity: Determine the precision and accuracy of the biosensor. Accomplishments: Repeatability studies showed that the SMART biosensor signal was highly repeatable. More extensive assays at various repeatability conditions are ongoing. Proposed Activity: Benchmark existing tests in the market that are used by the poultry industry. Accomplishments: The material cost of the SMART biosensor was estimated and compared to the price of commercially available tests. The analysis time of the SMART biosensor was compared to that of the commercially available tests. Proposed Activity: Assess the capabilities and constraints of potential users of the biosensor. Accomplishments: Two undergraduate students were asked to use the SMART biosensor in some experiments. User feedback showed that the biosensor was simple to use.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Caliskan-Aydogan O, Sharief S, and Alocilja EC. 2023. Rapid Isolation of Low-level Carbapenem-Resistant E. coli from Water and Foods Using Glycan-Coated Magnetic Nanoparticles, Biosensors 2023, 13(10), 902.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Caliskan-Aydogan O and Alocilja EC. 2023. A Review of Carbapenem Resistance in Enterobacterales and Its Detection Techniques. Microorganisms, 2023, 11(6), 1491.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Boodoo C, Dester E, David J, Patel V, Rabin KC, and Alocilja EC. 2023. Multi-Probe Nano-Genomic Biosensor to Detect S. aureus from Magnetically-Extracted Food Samples, Biosensors, accepted, in press. Biosensors 2023, 13(6), 608.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Sharief S, Caliskan-Aydogan O, Alocilja EC. 2023. Carbohydrate-coated nanoparticles for PCR-less genomic detection of Salmonella from fresh produce, Food Control, Vol. 150, 109770.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Boodoo C, Dester E, Sharief S, and Alocilja EC. 2023. Influence of Biological and Environmental Factors in the Extraction and Concentration of Foodborne Pathogens using Glycan-Coated Magnetic Nanoparticles, ?Journal of Food Protection, 86(4):100066.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Sharief SA, Caliskan-Aydogan O, and Alocilja EC. 2023. Carbohydrate-coated nanoparticles for point-of-use food contamination testing, Biosensors and Bioelectronics: X, 13 (2023), 100322, 9 pp.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Caliskan-Aydogan O, Sharief S, and Alocilja EC. 2023. Nanoparticle-Based Plasmonic Biosensor for the Unamplified Genomic Detection of Carbapenem-Resistant Bacteria, Diagnostics, 2023, 13(4), 656.
• Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Ghazy A, Nyarku R, Faraj R, Bentum K, Woube Y, Williams M, Alocilja E, and Abebe W. Gold Nanoparticle Colorimetric Detection of Escherichia coli, Salmonella enterica, Campylobacter jejuni, and Listeria monocytogenes from bovine fecal Samples, Journal of Microbiology Research, in review.

Progress 05/15/22 to 05/14/23

Outputs
Target Audience:During the reporting period (May 15, 2022 - May 14, 2023), we worked with the following industry groups: Michigan Turkey Producers Miller Poultry Alexander Strauch Consulting Comments from our stakeholders: "A rapid test for the industry is very much needed. The USDA continues to look forward to proposing changes in the salmonella standards and procedures associated with results. The timeliness in which processors and potentially growers can make pathogen determinations could be critical if the USDA makes its proposed changes." "Current and ongoing political discussions surrounding poultry-based foodborne illnesses from Salmonella include (1) Proposed regulatory frameworks that would increase Salmonella testing requirements, (2) Advocacy group pressure(s) for 'pre-harvest' Salmonella mitigation measures, and (3) Historical signaling from USDA's Tom Vilsack that he is amenable to points 1 & 2." "This article also made me think about our project and the value of cooperating with industry!" "I wonder if the pathogenic strains of Salmonella could be evaluated in poultry feed with our biosensors and subsequently impact the addition of a prebiotic of phage to address the internal gut performance and shedding. This area of research continues to be of utmost importance as FSIS moves forward with more regulation around it." Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training opportunities on this project were provided to: Three graduate students Six undergraduate research assistants Four visiting scholars How have the results been disseminated to communities of interest?Results from this project were disseminated through the following venues: Presentation at the 2022 meeting of the American Society of Agricultural and Biological Engineers, July 17-20, 2022 Presentation at the annual meeting of the Multistate NC-1194, Aug. 11-12, 2022 As an invited talk, the overall thrust of the project and preliminary results were presented at the FSIS Science and Technology Seminar Series on Nov. 16, 2022 What do you plan to do during the next reporting period to accomplish the goals?Plans for next year include: Use the MACE and biosensor assays to collect turkey and poultry samples from processing plants and farms. Validate the MACE and biosensing assays at the processing sites. Optimize the smartphone application for image display and analysis for the biosensing assay.

Impacts
What was accomplished under these goals? Year 1 Project Milestones and Accomplishments (May 15, 2022 - May 14, 2023) Proposed plan: Optimize enrichment assay, lab setting. Accomplishments: Magnetically activated cell enrichment (MACE) assay using our glycan-coated magnetic nanoparticles was optimized in large samples (100 mL or 25 g). Preliminary results showed that the assay for large samples could be completed in 15-30 min. The MACE assay was done on the following spiked samples: (1) ground meat and meat cuts, (2) carcass swabs from rehang, pre-chill, and post-chill, (3) cage swabs, (4) chiller water, and (5) mechanically separated meat. Preliminary results showed that the MACE assay concentrated the bacteria 1.5-5 times more than samples without MACE. The MACE assay was performed directly on poultry and bovine fecal samples. Preliminary results showed that the MACE assay concentrated the bacteria more than samples without MACE. Proposed plan: Optimize sample collection and sample processing, lab setting. Accomplishments: Samples were collected from the following sites in a turkey processing plant: cages, rehang, pre-chill, post-chill, chiller, and meat processing. Proposed plan: Optimize the biosensor, lab setting Accomplishments: The biosensor assay was performed on magnetically enriched cells without the need for culturing. The biosensor assay was performed on the following samples: (1) carcass swabs, (2) mechanically separated meat, and (3) poultry and bovine fecal samples. Preliminary results showed that the biosensor could detect Salmonella in these samples within 40 min. The biosensor results were confirmed using the plating method after 24 h of incubation. PCR was used to validate the biosensor results for S. Typhimurium extracted by the magnetic nanoparticles without any culturing. Proposed plan: Benchmark existing tests in the market that are used by the poultry industry. Accomplishments: Commercially available tests, such as Neogen's ANSR® for Salmonella and the culture method, were used at the turkey plant. ANSR takes 18 h of growth and 1 h of testing; culture takes 48 h of growth and plate incubation.

Publications