Recipient Organization
TUSKEGEE UNIVERSITY
(N/A)
TUSKEGEE,AL 36088
Performing Department
Microbiology
Non Technical Summary
This integrated project is designed to enhance the much-needed infrastructure and quality of Teaching, Research, and Extension at the Tuskegee University College of Veterinary Medicine (TUCVM), the only HBCU with a CVM. The proposed pre-harvest food 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. Graduate students will gain hands-on experience in these cutting-edge technologies to study the microbiome of cattle in the farms of small resource 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 to create 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 proposed extension activities. Workshops for farmers will focus on better herd health management practices to reduce shedding of pathogens such as Campylobacter, Salmonella, Listeria, and Shiga toxigenic E. coli. The project is a collaborative effort of a multidisciplinary team at TU, Michigan State University, and FDA Center for Veterinary Medicine, with expertise in the area of food 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, and Extension at the Tuskegee University College of Veterinary Medicine (TUCVM), the only HBCU with a CVM. The proposed pre-harvest food 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. Graduate students will gain hands-on experience in these cutting-edge technologies to study the microbiome of cattle in the farms of small resource 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 to create 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 proposed extension activities. Workshops for farmers will focus on better herd health management practices to reduce shedding of pathogens such as Campylobacter, Salmonella, Listeria, and Shiga toxigenic E. coli. The project is a collaborative effort of a multidisciplinary team at TU, Michigan State University, and FDA Center for Veterinary Medicine, with expertise in the area of food 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%
Goals / Objectives
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 safetyB. Enhance research infrastructure in food safetyStudy design, sampling & classification of animals into shedders, intermittent shedders and non-sheddersIdentification of priority four pathogensMultiple genes PCR array for specific identification of priority four pathogensMetagenomics study of shedders, and non-shedders of cattle in cow-calf farmsMultiplex nanoparticle-based DNA Biosensor C. Educate minority farmers of the black belt counties of Alabama on better herd health management system, to alleviate pre-harvest food 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 molecular genomic technologies. Provision of student experiential learning opportunities with innovative teaching methodologies to teach veterinary students and train graduate students interested in foodsafety.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 each farmer 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 reported prevalence (7.4%) and d is the desired precision (3%) [30]. To adjust for clustering, using the formula: DE=1+ p (m-1), were p is intra-class correlation coefficient (ICC), and m is the average size of the cluster, and DE is the design effect. Using a likely upper limit 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 the estimated 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 and repeatedly sampled every month for the first six months of year II of the project. Approximately 35 shedders and 15 non-shedders will be sampled. A super-shedder will be defined as an animal that sheds ≥4 log10 CFU g-1 and a heavy shedder as one shedding 3-4 log10 CFU g-1, shedders (<3 log10 CFU g-1), transient shedders (non-persistent shedders and non-shedders (no growth), as enumerated by direct fecal culture. Besides, shedders will be classified into two categories; 'persistent shedders,' if they shed the selected pathogens for three or more consecutive months and intermittent super-shedders or heavy shedders if they showed at least one shedding of4 log10 CFU g-1 or 3-4 log10 CFU g-1, respectively.b. Identification of priority four pathogensCampylobacter identification: Approximately 1g of feces will be diluted in 9ml of buffered peptone-water. A 400µl aliquot of the 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 10g of feces will be added to 90mL 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 will be aseptically transferred to 10mL of Listeria UVM II enrichment broth and incubated for another 24h at 30°C. Then, 100μL of the culture will be streaked on Modified Oxford agar plates (MOX) and incubated at 35°C for 48h. Black colonies with esculin hydrolysis 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, 10 g of the fecal samples will be mixed with 90mL of buffered peptone water and incubated at 37°C for 24h. Subsequently, selective enrichment will be performed by culturing 1mL of sample into 10mL of Rappaport-Vassiliadis medium. The culture will be 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 1g of each fecal sample will be mixed with 9ml of E.coli broth and incubated at 40ºC for 6 h. After incubation, separate broth aliquots will be subjected to immune-magnetic separation (IMS) using a King Fisher TM Flex Magnetic Particle Processor (Thermo Scientific, Waltham, MA) with anti-O157 Dynabeads (Invitrogen, Carlsbad, CA), and IMS beads for E.coli O26, O45, O103, O111, O121, and O145. Washed IMS beads will be spread onto Posse' differential agar and incubated for 18 h at 37ºC. Few colonies per mPosse' plate will be picked; on plates inoculated with O26, O45, O103, O111, or O157 IMS-treated cultures, red or blue-purple colonies will be picked and for O121 or O145 IMS-treated cultures, red or blue-purple, 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 common phenomenon of Campylobacter, Listeria, Salmonella, and STEC. Target genes such as omp50-p, mapA, ceuE, flaA for C.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 genes will be used for specific screening of O26, O45, O55, O103, O111, O121, O145, O157 and STEC-like organisms from all the 50 animals 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 metagenomics analysis using dry ice to BGI (BGI, Cambridge, MA). Metagenomics sequencing will be performed using BGISEQ-500 (BGI's DNBseq™ 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 DNA sequences 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 concentrate bacterial 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 involves combining (a) oligonucleotide probes specific to STEC, C.jejuni/coli, L.monocytogenes, and S.Typhimurium, (b) our functionalized 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 5min, anneal the probe at 55°C for 10 min, and then cool to room temperature. At this stage, the probes will anneal to the target DNA but will be free in solution if there are non-target DNA or no DNA. During the annealing, the GNP will self-assemble on the amine side of the probe through thiol-amine binding 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 the environment. During an annually organized workshop for farmers at the Center, students will take the leadership role in addressing evidence-based oration and presentations and possible mitigation strategies to reduce foodborne organisms shedding.