INTEGRATED APPROACH TO ADVANCE FOOD SAFETY TEACHING, RESEARCH & EXTENSION USING NANO-BIOSENSOR, GENOMICS, AND MICROBIOME AT TUSKEGEE UNIVERSITY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1026207
• Grant No.: 2021-38821-34710
• Cumulative Award Amt.: $399,195.00
• Proposal No.: 2020-10992
• Project Start Date: May 15, 2021
• Project End Date: May 14, 2026
• Grant Year: 2021
• Program Code: [EP]- Teaching Project

Recipient Organization
TUSKEGEE UNIVERSITY
(N/A)
TUSKEGEE,AL 36088

Performing Department
Pathobiology

Non Technical Summary
This integrated project is designed to enhance the much-needed infrastructure and quality of Teaching, Research, andExtension at the Tuskegee University College of Veterinary Medicine (TUCVM), the only HBCU with a CVM. The proposed preharvestfood safety focus is critical for training minority graduate students (MS and PhD) and veterinary students, with updated"Food Safety MBIO 512" course incorporating state-of-the-art microbiome, nano-biosensors, and genomics studies. Graduatestudents will gain hands-on experience in these cutting-edge technologies to study the microbiome of cattle in the farms of smallresource farmers in the "Black-Belt" region of West Alabama and categorize cows into super shedders, intermittent shedders,and non-shedders of selected foodborne pathogens. "Experiential Learning" activities for veterinary students are expected tocreate interest in careers related to food animal health & food safety. Keeping in view that interventions in the "Farm-To-Fork"continuum are needed to ensure safe foods, this project integrates teaching and research activities in conjunction with proposedextension activities. Workshops for farmers will focus on better herd health management practices to reduce shedding ofpathogens such as Campylobacter, Salmonella, Listeria, and Shiga toxigenic E. coli. The project is a collaborative effort of amultidisciplinary team at TU, Michigan State University, and FDA Center for Veterinary Medicine, with expertise in the area offood safety, genomics, microbiome, and nano-biotechnology. Resources acquired will significantly benefit the newly established"Center of Excellence for Food Animal Health & Food Safety" and the "Interdisciplinary Pathobiology PhD" program at TUCVM..This integrated project is designed to enhance the much-needed infrastructure and quality of Teaching, Research, andExtension at the Tuskegee University College of Veterinary Medicine (TUCVM), the only HBCU with a CVM. The proposed preharvestfood safety focus is critical for training minority graduate students (MS and PhD) and veterinary students, with updated"Food Safety MBIO 512" course incorporating state-of-the-art microbiome, nano-biosensors, and genomics studies. Graduatestudents will gain hands-on experience in these cutting-edge technologies to study the microbiome of cattle in the farms of smallresource farmers in the "Black-Belt" region of West Alabama and categorize cows into super shedders, intermittent shedders,and non-shedders of selected foodborne pathogens. "Experiential Learning" activities for veterinary students are expected tocreate interest in careers related to food animal health & food safety. Keeping in view that interventions in the "Farm-To-Fork"continuum are needed to ensure safe foods, this project integrates teaching and research activities in conjunction with proposedextension activities. Workshops for farmers will focus on better herd health management practices to reduce shedding ofpathogens such as Campylobacter, Salmonella, Listeria, and Shiga toxigenic E. coli. The project is a collaborative effort of amultidisciplinary team at TU, Michigan State University, and FDA Center for Veterinary Medicine, with expertise in the area offood safety, genomics, microbiome, and nano-biotechnology. Resources acquired will significantly benefit the newly established"Center of Excellence for Food Animal Health & Food Safety" and the "Interdisciplinary Pathobiology PhD" program at TUCVM.

Animal Health Component

25%

Research Effort Categories

Basic

25%

Applied

25%

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3310	1040	50%
903	3499	1103	50%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 903 - Communication, Education, and Information Delivery;

Subject Of Investigation
3499 - Dairy cattle, general/other; 3310 - Beef cattle, live animal;

Field Of Science
1040 - Molecular biology; 1103 - Other microbiology;

Keywords

pcr

cattle micrbiome

foodborne pathogens

foodsafety

integrated proposal

nanobiosensor

Goals / Objectives
A. Enhance capacity to teach Food Safety by incorporating novel contents such as Nano-biosensors, Microbiome, andMolecular technologies into the existing course of food safetyB. Enhance research infrastructure in food safety1. Study design, sampling & classification of animals into shedders, intermittent shedders and non-shedders2. Identification of priority four pathogens3. Multiple genes PCR array for specific identification of priority four pathogens4. Metagenomics study of shedders, and non-shedders of cattle in cow-calf farms5. Multiplex nanoparticle-based DNA BiosensorC. Educate minority farmers of the black belt counties of Alabama on better herd health management system, to alleviate preharvestfood contamination

Project Methods
Specific Objective 1: Enhance capacity to teach Food SafetyModification of the current Food Safety Course (MBIO523) to introduce latest nano-biotechnological, microbiome and moleculargenomic technologies. Provision of student experiential learning opportunities with innovative teaching methodologies to teachveterinary students and train graduate students interested in food safety.a. Study design, sampling & classification of animals into shedders, intermittent shedders and non-sheddersPhase I : Cross-sectional study. During the initial visit to the selected farms, a questionnaire will be administered to eachfarmer to obtain information on potential independent variables for detection of the four pathogens (dependent variables).Estimated sample size (no )=105 was determined using the following formula: {1.962 x Pexp x (1- Pexp )}/d2 where Pexp is reportedprevalence (7.4 %) and d is the desired precision (3%) [30]. To adjust for clustering, using the formula: DE=1+ p (m-1), were p isintra-class correlation coefficient (ICC), and m is the average size of the cluster, and DE is the design effect. Using a likely upperlimit for ICC of 0.2 and an average cluster size of 11 (11 farms selected), DE = 1 + 0.2 (11 - 1) = 1 + 2 = 3. Multiplying DE by theestimated sample size = 3 x 105 = 315. Therefore, for the study, we plan to collect a minimum of 320 adult cattle.Phase II: Longitudinal study : 50 cattle will be randomly selected and tagged during the first visit of the longitudinal study andrepeatedly sampled every month for the first six months of year II of the project. Approximately 35 shedders and 15 nonshedderswill be sampled. A super-shedder will be defined as an animal that sheds ≥4 log10 CFU g-1 and a heavy shedder asone shedding 3-4 log10 CFU g-1 , shedders (<3 log10 CFU g-1 ), transient shedders (non-persistent shedders and non-shedders (nogrowth), as enumerated by direct fecal culture. Besides, shedders will be classified into two categories; 'persistent shedders,' ifthey shed the selected pathogens for three or more consecutive months and intermittent super-shedders or heavy shedders ifthey showed at least one shedding of 4 log10 CFU g-1 or 3-4 log10 CFU g-1 , respectively.b. Identification of priority four pathogensCampylobacter identification : Approximately 1 g of feces will be diluted in 9 ml of buffered peptone-water. A 400 μl aliquot ofthe fecal suspension will be plated to the surface of modified blood-free charcoal, cefoperazone deoxycholate agar (CM 739;Oxoid, Ogdensburg, NY).Listeria isolation from feces: Approximately 10 g of feces will be added to 90 mL of Listeria UVM I enrichment broth (Oxoid®),homogenized for two minutes in a stomacher (Seward®) and will be incubated at 30°C for 24 h. Then, 100μL of the culture willbe aseptically transferred to 10 mL of Listeria UVM II enrichment broth and incubated for another 24 h at 30°C. Then, 100μL ofthe culture will be streaked on Modified Oxford agar plates (MOX) and incubated at 35°C for 48 h. Black colonies with esculinhydrolysis representing Listeria phenotype will be analyzed for further species identification using our omni log PM.Salmonella identification: We will follow the ISO 6579 standard for the identification of Salmonella spp. For pre-enrichment, 10g of the fecal samples will be mixed with 90 mL of buffered peptone water and incubated at 37°C for 24 h. Subsequently,selective enrichment will be performed by culturing 1 mL of sample into 10 mL of Rappaport-Vassiliadis medium. The culture willbe incubated at 42°C for 16-24 h. Samples will be inoculated onto xylose lysine deoxycholate (XLD) and brilliant green (BG)agars.STEC identification: Approximately 1 g of each fecal sample will be mixed with 9 ml of E. coli broth and incubated at 40ºC for 6h. After incubation, separate broth aliquots will be subjected to immune-magnetic separation (IMS) using a King Fisher TM FLEXMagnetic Particle Processor (Thermo Scientific, Waltham, MA) with anti-O157 Dynabeads (Invitrogen, Carlsbad, CA), and IMSbeads for E. coli O26, O45, O103, O111, O121, and O145. Washed IMS beads will be spread onto Posse' differential agar andincubated for 18 h at 37ºC. Few colonies per mPosse' plate will be picked; on plates inoculated with O26, O45, O103, O111, orO157 IMS-treated cultures, red or blue-purple colonies will be picked and for O121 or O145 IMS-treated cultures, red or bluepurple,and green colonies will be picked.c. Multiple genes PCR array for specific identification of priority four pathogensThis array will also be used to rule out those animals shedding viable but non-culturable pathogens, which is a very commonphenomenon of Campylobacter, Listeria, Salmonella, and STEC. Target genes such as omp50-p, mapA, ceuE, flaA forC. jejuni/coli, and mo1118, lmo0737, ORF2110, ORF2819 and prs but also to profile serotypes of (1/2a, 1/2b, 1/2c and 4b),including inlA will be used for all isolates phenotypically identified as Listeria. Several genes including, stx1 , stx2 , eae, ehxA,rfbeEO157 , aggR, wzy, wbgN, wdbI and aggR. Virulence genes stx1, stx2, eae, and ehxA and aggR, wzy, wbgN and wdbI geneswill be used for specific screening of O26, O45, O55, O103, O111, O121, O145, O157 and STEC-like organisms from all the 50animals selected in the longitudinal study.d. Metagenomics study of shedders, and non-shedders of cattle in cow-calf farmsMetagenomic sequencing and analysis. Fecal whole genomic DNA will be extracted using Qiagen QIAmp DNA stool Minikit(Qiagen, Germantown, MD), the quality of DNA will be checked using Nanodrop2000c (Thermo Fisher Scientific, Waltham, MA).The required DNA concentration of ≥100ng will be resuspended in nuclease-free water and will be sent for metagenomicsanalysis using dry ice to BGI (BGI, Cambridge, MA). Metagenomics sequencing will be performed using BGISEQ-500 (BGI'sDNBseq™ technology). Base calls generated by the DNBseq System will be converted to FASTQ files for further analysis.Reads will be de-multiplexed and analyzed for quality using FastQC (Babraham Bioinformatics). Low quality and host DNAsequences will be removed from the raw metagenomic sequences using Trimmomatic and Bowtie2, respectively.e. Multiplex nanoparticle-based DNA BiosensorStudy 1.1. DNA extraction. Our own functionalized magnetic nanoparticles (MNP) will be used to isolate and concentratebacterial cells from farm samples without centrifugation.Study 1.2. GNP-DNA Detection. The multiplex genomic DNA detection is a simple two-step process. The first step involvescombining (a) oligonucleotide probes specific to STEC, C. jejuni/coli, L. monocytogenes, and S. Typhimurium, (b) ourfunctionalized 30-nm gold nanoparticles, and (c) extracted DNA (target and non-target) in their respective microcentrifuge tubes.The tubes will be placed in the thermocycler to denature the genomic DNA at 95 °C for 5 min, anneal the probe at 55 °C for 10min, and then cool to room temperature. At this stage, the probes will anneal to the target DNA but will be free in solution if thereare non-target DNA or no DNA. During the annealing, the GNP will self-assemble on the amine side of the probe through thiolaminebinding reaction, generating the GNP-probe-DNA nanostructures.Specific objective 3: Educate minority farmers of the black belt counties of AlabamaIdentification of the herd management practices will help to understand which practices promote contamination of theenvironment. During an annually organized workshop for farmers at the Center, students will take the leadership role inaddressing evidence-based oration and presentations and possible mitigation strategies to reduce foodborne organismsshedding.

Progress 05/15/24 to 05/14/25

Outputs
Target Audience: During the reporting period (May 15, 2024 - May 14, 2025), we worked with the following groups: 1.Graduate students and DVM students 2.Food Security Solution (FSS), a Company Licenced our four-pathogen detection platform including multiple Salmonella serovars, Campylobacter, STEC (hence forward named GenoPathX Abbreviated as GPX) 3.Local farmers of Alabama 4.Graduate students in the Material Science Engineering department in Tuskegee University 5.Faculty and interns of Large Animal Ambulatory Services at Tuskegee University, College of Veterinary Medicine 6.The USDA/ARS laboratory at Athens, GA 7.Auburn University College of Veterinary Medicine 8.State Diagnostic lab at Auburn, AL? Changes/Problems:NA What opportunities for training and professional development has the project provided?Training opportunities on this project were provided to 6 graduate students, 2 DVM Summerstudents, One student was able to defend his MSc and was able to be accepted as a PhD Candidate, the second MSc Student under this project is doing doual MSc/DVM programACCOMPLISHED How have the results been disseminated to communities of interest?Results were presented at Different conferences To local farmers: Professional Agricultural Workers Conference (PAWC) at Montgomery, Alabama, November 17, 2024, To Scientific Community : Conference of Research workers in Animal Diseases(CRWAD)Chicago, Illinois, January 20, 2025 American Society for Microbiology (ASM) Microbe Annual Conference: Atlanta, Georgia, June 14, 2024 International Association for Food Protection (IAFP) Annual Conference, Long Beach, California, July 13, 2024 To faculty, staff and Graduate students at Tuskegee Ubiversity: Tuskegee University College of Veterinary Medicine, Annual Phi Zeta Research Day, Tuskegee, Alabama, September 17, 2024 What do you plan to do during the next reporting period to accomplish the goals? Publish more scientific papers especially the Metagenomics study of shedders, and non-shedders of cattle in cow-calf farms Dissiminate our findings through presentation at national and International conferences Utilize our oun multiplex GPX assay for identification of priority four pathogens from food sources, processing plants and animal samples

Impacts
What was accomplished under these goals? A. Enhance capacity to teach Food Safety by incorporating novel contents such as Nano-biosensors, Microbiome, and Molecular technologies into the existing course of food safety ACCOMPLISHED B. Enhance research infrastructure in food safety 1. Study design, sampling & classification of animals into shedders, intermittent shedders and non-shedders 2. Identification of priority four pathogens 3. Multiple genes PCR array for specific identification of priority four pathogens ACCOMPLISHED 4. Metagenomics study of shedders, and non-shedders of cattle in cow-calf farms 5. Multiplex nanoparticle-based DNA Biosensor PARTIALLY ACCOMPLISHED C. Educate minority farmers of the black belt counties of Alabama on better herd health management system, to alleviate preharvest food contamination ACCOMPLISHED

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Name of the presenter: Rejoice Nyarku Title of the presentation: PARTNERSHIP: Development of a Nanoparticle-enabled Enrichment and DNA-Biosensing for Onsite Monitoring of Multiple Foodborne Pathogens in Large Samples: Progress Report 2023-2024. Name of the organization/scientific conference: International Association for Food Protection (IAFP) Annual Conference City, State, and Date of the presentation: Long Beach, California, July 13, 2024
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Name of the presenter: Rejoice Nyarku Title of the presentation: Integrating GenoPATHX assay and Nano Biosensor Technologies for Comprehensive Analysis of Salmonella Serovar Dynamics in Poultry Products. Name of the organization/scientific conference: American Society for Microbiology (ASM) Microbe Annual Conference City, State, and Date of the presentation: Atlanta, Georgia, June 14, 2024
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: In-silico Detection of Gene-Specific Markers for Campylobacter Species Utilizing Pangenome and Core Genome Analysis Authors: Emmanuel Kuufire1, Kingsley Bentum1, Rejoice Nyarku1, Viona Osei1, Samuel Temesgen1, Woubit Abebe1* Name of the organization/scientific conference: Tuskegee University College of Veterinary Medicine, Annual Phi Zeta Research Day City, State, and Date of the presentation: Tuskegee, Alabama, September 17, 2024
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: In-silico Detection of Gene-Specific Markers for Campylobacter Species Utilizing Pangenome and Core Genome Analysis Authors: Emmanuel Kuufire1, Kingsley Bentum1, Rejoice Nyarku1, Viona Osei1, Samuel Temesgen1, Woubit Abebe1* Name of the organization/scientific conference: Professional Agricultural Workers Conference (PAWC) City, State, and Date of the presentation: Montgomery, Alabama, November 17, 2024
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: In-silico Detection of Gene-Specific Markers for Campylobacter Species Utilizing Pangenome and Core Genome Analysis Authors: Emmanuel Kuufire1, Kingsley Bentum1, Rejoice Nyarku1, Viona Osei1, Samuel Temesgen1, Woubit Abebe1* Name of the organization/scientific conference: Conference of Research workers in Animal Diseases (CRWAD) City, State, and Date of the presentation: Chicago, Illinois, January 20, 2025
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: " Kingsley Bentum, Emmanuel Kuufire, Rejoice Nyarku, Viona Osei, Temesgen Samuel, Woubit Abebe. Genomic comparison of Salmonella Typhimurium and Salmonella Dublin isolates from bovine sources in the U.S. Conference of Research Workers in Animal Diseases (CRWAD) 2025, January 18-21, 2025, Chicago, IL, Poster Presentation
• Type: Other Journal Articles Status: Under Review Year Published: 2025 Citation: In-silico Detection of Gene-Specific Markers for Campylobacter Species Utilizing Pangenome and Core Genome Analysis Authors: Emmanuel Kuufire, Kingsley Bentum, Rejoice Nyarku, Viona Osei, Samuel Temesgen, Woubit Abebe* MDPI Pathogens
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Name of the presenter: Rejoice Nyarku Title of the presentation: Validation of a GenoPATHX qPCR assay for rapid and sensitive detection of Salmonella enterica serovars in Poultry: Implications for enhanced food safety and public health. Name of the organization/scientific conference: Tuskegee University College of Veterinary Medicine, Annual Phi Zeta Research Day City, State, and Date of the presentation: Tuskegee, Alabama, September 17, 2024
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Ghazy, A.; Nyarku, R.; Faraj, R.; Bentum, K.; Woube, Y.; Williams, M.; Alocilja, E.; Abebe, W. Gold Nanoparticle-Based Plasmonic Detection of Escherichia coli, Salmonella enterica, Campylobacter jejuni, and Listeria monocytogenes from Bovine Fecal Samples. Microorganisms 2024, 12, 1069. https://doi.org/10.3390/microorganisms12061069
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Bentum, K.E.; Kuufire, E.; Nyarku, R.; Osei, V.; Price, S.; Bourassa, D.; Samuel, T.; Jackson, C.R.; Abebe, W. Salmonellosis in Cattle: Sources and Risk of Infection, Control, and Prevention. Zoonotic Dis. 2025, 5, 4. https://doi.org/10.3390/zoonoticdis5010004
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Faraj, R.; Ramadan, H.; Bentum, K.E.; Alkaraghulli, B.; Woube, Y.; Hassan, Z.; Samuel, T.; Adesiyun, A.; Jackson, C.R.; Abebe, W. Antimicrobial Resistance, Virulence Gene Profiling, and Spa Typing of Staphylococcus aureus Isolated from Retail Chicken Meat in Alabama, USA. Pathogens 2025, 14, 107. https://doi.org/10.3390/pathogens14020107
• Type: Other Journal Articles Status: Under Review Year Published: 2025 Citation: Hydrogen Sulfide negative Salmonella and their implication for standard culture-based identification. Under Review: Journal of Food Protection
• Type: Other Journal Articles Status: Under Review Year Published: 2025 Citation: First report of the isolation of an H2S-negative Salmonella Typhimurium i-4-1,2 from cattle in rural Alabama-U.S in an investigation into fecal shedding by cow-calf herds in the area. Under Review Frontiers in Veterinary Science.
• Type: Other Journal Articles Status: Under Review Year Published: 2025 Citation: Comparative genomic profiles of Salmonella Typhimurium and Salmonella Dublin bovine isolates from the U.S. indicate possible factors associated with host adaptation of Salmonella Dublin in the region. Under Review MDPI Microorganisms

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

Outputs
Target Audience: During the reporting period (May 15, 2023 - May 14, 2024), we worked with the following groups: 1.West Alabama rural farmer community 2.DVM students 3.Graduate students 4.Turkey and poultry Farms for testing validatingSalmonellaserovar detection 5.Graduate students and DVM students 6.Food Supply Security, a Company interested in licensing our priority four pathogen detection platform including multiple Salmonella serovars, Campylobacter, STEC (hence forward named GenoPathX)? Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Fellowship received based on their research output of through this grant a.Kingsley Bentum received FSIS fellowship for one year as a result of his research study on Salmonella and campylobacter from cow-calf farming system of AL with this grant work, b.Rejoice Nyraku received AAUW International Doctoral Degree Fellowships in the amount of $ 25000, for her work on Salmonella quantitation and novel target development for specific serovar detection. How have the results been disseminated to communities of interest?Magnetic Nanoparticles, a Potential Biosensor to Aid in the Rapid Molecular Detection ofSalmonellaTyphimurium at Sub-Infectious Dose Levels. Kingsley Bentum, Ahmed. Ghazy Yilkal Woube, Rawah Faraj, Tyric James, Temesgen Samuel, Evangelyn Alocija, Woubit Abebe. IAFP meeting 2023, July 13-17 Toronto, Canada Exploring Qualitative and Quantitative presence of Salmonella Serovars from Poultry products using Genovar and Nano Biosensor Technologies. Kierney Burks1, Rejoice Nyarku1, Kingsley Bentum1, Dr. Rawah Faraj1, Dr. Alocilja Evangelyn2, Dr. Woubit Abebe*1.National veterinary Scholars symposium. San Juan, Puerto Rico, August 2-6th, 2023. And National Phi Zeta Research Day September 2023, Tuskegee AL Prevalence, virulence, molecular characterization, and phylogenetic tree analysis ofhlyAgene expression ofListeria monocytogenesisolated from cattle fecal samples from the central Area of Alabama.Sabria Wooten1, Woubit Abebe1, Rawah Faraj1.National veterinary Scholars symposium. San Juan, Puerto Rico, August 2-6th, 2023 and National Phi Zeta Research Day September 2023, Tuskegee AL Investigating the molecular mechanism of antibiotic resistance in salmonella within alternative poultry feeding approaches. Nyarku, Rejoice, Kingsley Bentum, Rawah Faraj, and Woubit Abebe.Carver Integrative Sustainability Center (CISC) HBCU Graduate Fellowshipprogram Professional Agricultural Workers Conference (PAWC), October 29-31, 2023,Montgomery, AL What do you plan to do during the next reporting period to accomplish the goals? Improve the current targets with further pangenome analysis and search to arrive at more throughput and robust targets to be used for biosensor Train more graduate and summer DVM research students Present at national and international meetings of our on-going findings Publish additional papers which are in pipeline for STEC organisms Perhaps complete the whole project in the coming year.

Impacts
What was accomplished under these goals? 1.Sensitivity and specificity were tested using multiple fecal samples and using artificially inoculated fecal samples to identifySalmonellaand other priority four pathogens from a complex matrix 2.Graduate and DVM students were trained 3.Communicated with farmers types of management practices to reduce shedding of priority four pathogens, this information is part of the points to be discussed on our upcoming papers on STEC. 4.A graduate student was hired to investigate molecular regions from the genomes of multiple salmonella to help expand the existing Salmonella platform that detects 30 serovars. 5.Course deployed along with the course VMED 872, where students introduced the concepts of metagenomics microbiome and nano-biosensor applications in the diagnosis of priority four pathogens 6.Completed metagenomics study with collaboration of FDA, we performed deep MiSeq and Data is still under analysis using GalaxyTracker, we also hope the information from this data will be one of our upcoming publication.?

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Gold Nanoparticle Colorimetric Detection of Escherichia coli, Salmonella enterica, Campylobacter jejuni, and Listeria monocytogenes from bovine fecal Samples. Ahmed Ghazy 1,2,3, Rejoice Nyarku 3, Rawah Faraj3, Kingsley Bentum3, Yilkal Woube3, McCoy Williams3, Evangelyn Alocilja4 and Woubit Abebe 3,* Reviewers' comment addressed, and the paper was resubmitted for consideration in publication in the Journal of Microbiology Research. Geographical Distribution of Salmonella isolated from food animal products in the Southeastern United States from 2019-2022. Kingsley Bentuma, Rejoice Nyarkua, Labeed Ben-Ghalyb, Johnette Brown-Silvab, Evelyn Crishb, Lisa Jacobsenb, Glenn Tillmanb, Aphrodite Dourisb, Mustafa Simmonsb, Temesgen Samuela, Isabel Wallsb, Woubit. Abebe*a. Submitted to Journal of Food Protection 2024

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

Outputs
Target Audience:-Local farmers of the Blackbelt region of the state of Alabama, -Graduate Students of the College Of Veterinary Medicine, TU -DVM students of theCollege Of Veterinary Medicine, TU, and Visiting Scholars Changes/Problems:Major problems, Due to major heavy rain and lightning,we had in the state of AL, we faced some challenges as our Freezers and Fridges went down. Asa result, we are facing a situation where we have to replace sample storage freezers and Fridges. This is something we plan on discussing with the program director as we move forward with the project work and training and see if we can find some solution. The initial plan of Sampling 350 animal samples became impossible due to so many unfathomable reasons. So we are now remaining with 200 heads of animal samples. What opportunities for training and professional development has the project provided?This Project tremendously improved our IDBP graduate students and DVM students' summer training opportunities As of now, we have 2 Masters students in Master of Science in Veterinary medicine students with tuition included 4 PhD Students 1 Graduate Fellowship, who all received training and we plan on continuing toexpand engaging 2 more DVM students this coming Summer How have the results been disseminated to communities of interest? So far we have participated inProfessional Agricultural Workers Conference (PAWC) in Montgomery, AL2022, where we discussed and shared our preliminary findings. We also met with local integrators and poultry producers interested in Salmonella and Campylobacter work of this project, We also plan on publishing our findings as we complete the work two manuscripts are currently in the pipeline of completion and submission What do you plan to do during the next reporting period to accomplish the goals?Definitely, the next reporting period will mainly focus on Manuscript preparation proposed course delivery Metagenomic analysis of the fecal microbiome and defining possible probiotic cocktail for mitigating shedding of priority four pathogens The year of research has been fairly good so far except for the major challenge of not getting cooperating farmers anymore for sampling. Nevertheless, within the period under review, a total of 200 fecal samples were collected from twenty-seven farms

Impacts
What was accomplished under these goals? A. Enhance capacity to teach Food Safety by incorporating novel contents such as Nano-biosensors, Microbiome, and Molecular technologies into the existing course of food safety B. Enhance research infrastructure in food safety 1. Study design, sampling & classification of animals into shedders, intermittent shedders and non-shedders 2. Identification of priority four pathogens 3. Multiple genes PCR array for specific identification of priority four pathogens Accomplished 4. Metagenomics study of shedders, and non-shedders of cattle in cow-calf farms This is still underway due to some instrument obstacles we faced... We are able to perform this during the summer break with training included for DVM students 5. Multiplex nanoparticle-based DNA Biosensor Accomplished and the manuscript is already drafted and ready for submission after the inclusion of minor experiments C. Educate minority farmers of the black belt counties of Alabama on better herd health management systems, to alleviate preharvest food contamination We had our first meeting with farmers and stakeholders at the Professional Agricultural Workers Conference (PAWC) in Montgomery, AL2022, where we discussed and shared our preliminary findings.

Publications

Progress 05/15/21 to 05/14/22

Outputs
Target Audience:Farmers were the targeted audience in the past year's reporting period, as they have worked closely with our graduate students. We participated in theAnnual Farmers Conferences at Tuskegee University (Farmers Conference) to explain the objective of our work. Changes/Problems:We faced major setbacks with project personnel: In the original project plan, Dr. Ricardo Bridges and Dr. Kenneth Newkirk were tasked and agreed to participate in fecal and environmental sample collection from cattle farms from different counties of Alabama as participating members of the project. However, Dr. Bridges has retired and Dr. Newkirk was regrettably deceased. Because the project has to continue, we were looking for individuals to assist us with a knowledge of cattle farm operations and locations in the various counties. this together with the pandemic has halted all DVM students' travel, as a result, we lost ourlead person working with farmers and coordinating invitations of farmers for the annual workshop and forum at the Center. We then reached out to Mr. Russell Johnson, Sr, CAENS, George Washington Carver Agricultural Research Station, Farm Manager. Mr. Johnson was finding some time at the side to assist us during his regular duties. We made use of his expertise in handling cattle, his knowledge of the local farming community, and his record of helping undergraduate and graduate students in animal and poultry science. In addition, our longtime advisor and expert, Dr Gopal Reddy retired, which may have caused a slight delay in the deployment of the course, luckily we plan on achieving this goal by the next Spring semester. We also faced some challenges: Due to COVID and lack of many personnel themajor set back had been the delay in getting the needed materials to work with as the pandemic had led to a huge backlog of orders with manufacturers. What opportunities for training and professional development has the project provided?Our collaboration with MSU and FDA has provided significant training opportunities for our graduate and Summer DVM research students. Primarily graduate students have acquired knowledge in microbial data analysis and sequencing. In addition, DVM and graduate students can now use Magnetic and Gold nanoparticles as a means of pathogen extraction and Gold biosensor based specific detection of priority four pathogens described in this project (Listera monocytogenes, STEC Escherichia coli, Salmonella serovars, and Campylobacter jejuni and coli). In addition, our students now have state-of-the-art research equipment, such as a sequencing machine and Phenotypic microarray, which they are learning and incorporating into their respective research endeavors.? How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?1= Finalize sampling as of now we still have 200 animals to sample, and some animals will be selected for a longitudinal study to assess shedders, intermittent shedders, and non-shedders 2= Prepare and deploy the Food Safety course (MBIO523) 3= Develop PCR array for simultaneous detection of Priority four pathogens 4= Develop multiple nano-biosensor 5= Hire one more graduate student 6= Perform a metagenomic study to identify cattle microbiome influence of priority four shredders 7= Interact with local farmers and advice on what our results show to help curb shedding and preharvest food contamination

Impacts
What was accomplished under these goals? Project Goal A is underway with graduate students, including 2 Ph.D.and 1 Master of Science in Veterinary medicine have now been fully engaged in the project research. The project also hired 2 DVM students to participate in the project's research in quantitativeSalmonella work and the development of a nano biosensor for rapid detection ofListera monocytogenes. The course preparation is underway and projected forthe Spring 2023 semester. In addition, the PI has multiple courses to teach during the Fall semester. Project Goal B. Has been achieved now we are independently able to perform genotypic and phenotypic characterization of priority four pathogens, able to perform in-house sequencing and training, able to use Gold and magnetic nanoparticles for specific identification of priority four pathogens B.1.During the initial phase, required equipment and materials were obtained, and protocols were developed. In the study latest bacteriological and molecular methods were used. A considerable period of time, resources, effort, and time was taken to optimize the protocols. A number of protocols were then developed and optimized. B.2. STEC: A study was initiated to study Shiga toxin-producing Escherichia coli in Alabama in cow-calf operations. The objectives were to isolate and identify O157 and non-O157 strains, determine prevalence and risk factors, and assess the effect of virulence factors on cattle gut cellular immunity. The study focuses on O157 and the top seven non-O157 strains, which are O26, O45, O103, O104, O111, O121, and O145.Up to the first week of June 9, 2022, 19 farms were visited. From the farms, 244 fecal samples and 47 environmental samples were collected. A questionnaire data consisting of risk factors for infection were collected to be used for epidemiological analysis. Table 1. Summary of activities conducted before 6/9/2022. Farms Pens Fecal samples collected Environmental samples stx gene screening Questionnaire administered 19 25 244 47 291 fecal and environmental samples 19 Isolation and identification of samples from the first three farms were conducted. The prevalence of stx gene was 13%. In addition, O157, O26, O104, and O111 were identified. O104 strain was identified for the first time from Alabama. Overall from completed data stx2 is more common than stx1. In this study, at present, the occurrence of Shiga toxin-producing Escherichia coli of both O157 and non-O157 strains is observed to be high in Alabama cow-calf operations. B.2. Salmonella and campylobacter:Salmonella spp and Campylobacter spp (specifically Campylobacter coli and Campylobacter jejuni) Screening of the collected fecal samples for the above-mentioned pathogens has steadily been progressive. Screening primarily applies culture and molecular methods (PCR) to determine the presence or absence of the pathogens. As of the time of this report, a total of 90 samples from twelve (12) different farms have been screened and the results are summarized in the table below. FARM Total Number Positive C.jejuni Samples Positive C.coli Samples Positive Salmonella Samples Farm 14 10 7 6 9 Farm 5 9 5 5 7 Farm 15 3 0 1 3 Farm 10 7 2 1 7 Farm 13 10 4 5 10 Farm 8 4 1 2 4 Farm 9 8 0 0 8 Farm 11 8 5 6 8 Pen 6 16 2 8 14 Pen 4 4 2 0 4 Pen 5 6 1 1 6 Pen 3 5 2 2 5 Prevalence with respect to total samples screened 90 31(34%) 37 (41%) 85(94%) B.2. Listeria monocytogenes: Screening of the collected fecal samples for the Listeria monocytogenesstarted in November 2021. That includes isolation and identification of L. monocytogenes by different bacteriological and molecular techniques. Farm Number of total samples Positive L. monocytogenes Negative Prevalence of Contamination Farm 4 32 12 20 37.5% Farm 5 9 7 2 77.7% Farm 6 7 4 3 57.14% Farm 7 10 4 6 40% Farm 8 4 None 4 0% Farm 9 8 1 7 12.5% (need more work) Farm 10 7 2 5 28.6% Farm 11 8 3 5 37.5% (need more work) Farm 12 8 2 6 25% (need more work) Farm 13 10 2 8 20% Farm 14 10 2 8 20% Farm 15 3 0 3 0% Farm 16 10 1 9 10% Farm 17 11 5 6 45.45% Farm 18 8 4 4 50% Farm 19 15 2 13 13.3% Total Number 160 51 109 Total: 31.875 % B.3. Multiple genes PCR array for specific identification of priority four pathogens: Target-specific Primers are already developed and validated for all these pathogens includingEscherichia coli O157:H7,and O26, O45, O103, O104, O111, O121, and O145commonly known as non-O157 STEC. for Salmonella serotypes, Listeria monocytogenes andCampylobacter jejuni and coli. B.4. Metagenomics study of shedders, and non-shedders of cattle in cow-calf farms: genomic DNA from all fecal samples so far collected is isolated and stored at -20OC awaiting the completion of sampling. Once sampling is done on a total of 350 samples metagenomics study will be performed from shedders, non-shedders, and intermittent shedders of priority four pathogens B.5. Multiplex nanoparticle-based DNA Biosensor: Target Biosensors are developed and are ready to be used for Goldnanoparticle pathogen detection, preliminary work has already been conducted on Listera monocytogens and the result is promising. C. Educate minority farmers of the black belt counties of Alabama on better herd health management system, to alleviate preharvestfood contamination Not done yet,?

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