Source: TENNESSEE STATE UNIVERSITY submitted to NRP
PROFESSIONAL DEVELOPMENT IN MOLECULAR APPROACHES FOR TICK-BORNE PATHOGEN RESEARCH IN TERRESTRIAL ECOSYSTEMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1021785
Grant No.
2020-38821-31111
Cumulative Award Amt.
$97,294.00
Proposal No.
2019-03344
Multistate No.
(N/A)
Project Start Date
Jun 1, 2020
Project End Date
Dec 31, 2023
Grant Year
2020
Program Code
[EQ]- Research Project
Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209
Performing Department
Ag. & Env. Sciences
Non Technical Summary
The proposed Professional Development Grant will provide multiple environmental and social gains. The most important aspect of this project will be our ability to understand the prevalence of tick-borne pathogens and how these patterns change based on host species. These prevalence data will be most important for informing the natural resources and environmental sciences workforce along with the general public of the potential disease risks associated with outdoor work and recreational activities that co-occur in habitats where ticks are common. These data will be directly beneficial to maintenance of human health through increased knowledge of disease prevalence.Data acquired during this research will serve as preliminary data to acquire additional funding from other funding agencies. These continuation funds will permit opportunities to recruit additional graduate students and conduct supplementary projects to explore additional research questions related to wildlife conservation and disease ecology. Environmental benefits from this research include advancements in our understanding of how host type influences disease prevalence and associated vector microbial diversity. Although this research will occur locally in the southeastern United States, results are transferable across a variety of forest types and wildlife communities throughout the United States and globally.
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1363120111060%
1350613113040%
Knowledge Area
135 - Aquatic and Terrestrial Wildlife; 136 - Conservation of Biological Diversity;

Subject Of Investigation
3120 - Spiders, mites, ticks, and other arthropods; 0613 - Mixed conifer-broadleaf forests;

Field Of Science
1130 - Entomology and acarology; 1110 - Parasitology;
Goals / Objectives
This Professional Development Grantl seeks to increase capacity for the PI to work collaboratively with experts at Middle Tennessee University and the Tennessee Vector Borne Disease clinic to conduct laboratory assessment of tick-borne pathogen prevalence via quantitative Polymerase Chain Reaction (qPCR) and 16s amplicon sequencing approaches. In addition, this grant will provide additional capacity to mentor undergraduate and graduate students and for the development of new course material related to disease ecology and monitoring in the Department of Agricultural and Environmental Sciences at TSU. The outcomes of this grant are aligned with NIFA Priority Area of Environmental Sciences and will address emerging need of understanding the importance of ticks as zoonotic disease vectors, which is directly applicable to improving human health and well-being, along with safety of the natural resources workforce and general stakeholders that utilize natural landscapes for work and recreation. Over the course of the study, we will aim for thefollowing objectives:Objective 1: Utilize qPCR analysis and 16s amplicon sequencing approaches to evaluate microbial composition of ticks collected from a variety of forested environments and vertebrate hostsObjective 2: Develop and update course materials focused on disease ecology and wildlife disease for the Ecosystem Science and Management Course (AGSC 5510/7510) at TSUObjective 3: Planning for future collaborations, proposal development, and course instruction
Project Methods
Ticks collected from a long-term project in the William B. Bankhead National Forest, Alabama will be utilized during the proposed study. Collectively, the Wildlife Ecology Lab has accrued over 3,000 nymph and adult tick specimens from a variety of differently-managed forest stands and vertebrate host species. Sampled ticks will be identified to species using appropriate species identification keys. Adult ticks will be assigned to individual vials, whereas nymphs will be combined into three individuals per vial based on collection location. Prior to DNA extraction, each nymph adult tick will be dissected into half and four equal sections, respectively. We will place each tick tissue sample in a Qiagen Power Bead Tube and each sample will be flash frozen in liquid nitrogen for 10 minutes. We will add 500 μL of Eagle's Minimum Essential Media to each tube and will process the sample using a bead shaker for 5 minutes. We will centrifuge samples and extract genomic DNA from tick samples using DNEasy PowerSoil kits per the manufacturer's instructions. Extracted DNA will be stored in a -80? C freezer until further analysis.We will work with Dr. Abelardo Moncayo at the Tennessee Department of Health to screen for the presence of select tick-borne pathogens, including Erlichia sp., Rickettsia sp., Borrelia sp., and Anaplasma via qPCR known to cause disease in humans and wildlife.Our preliminary data suggests that at least three pathogens occur in ticks sampled from our study sites and these results differ by host species or environment. We will use qPCR to confirm pathogen presence/absence and relative pathogen load in approximately 2000 tick specimens collected previously from variety of vertebrate wildlife, including small mammals, lizards, and feral hogs, along with ticks collected during standardized tick drag surveys. For all qPCR assays, we will test each tick sample in triplicate and will use the QuantStudio 5 thermocycler platform. We will include a positive control of each focal pathogen and a negative water sample during each qPCR run. We will quantify relative pathogen load via Critical Threshold scores which will be based on 10-fold serial DNA dilutions.We will select a stratified sub-sample (maximum of 400 samples) of ticks from each of the focal wildlife hosts (small mammal, lizard, feral hog, and tick drag sample) confirmed as "positives" and "negatives" via qPCR for the targeted pathogens described above for further examination of microbial community composition using a 16s microbiome sequencing approach. We will work with Dr. Donald Walker (Middle Tennessee State University) to complete 16s rDNA sequence analysis on the Illumina platform). Bioinformatic preparation of the resulting sequence data will be performed in the program MOTHUR for 16S sequences. After assignment of operational taxonomic units (OTUs), we will calculate both alpha and beta diversity statistics. We will further evaluate 16s sequence diversity by calculating beta diversity via Bray-Curtis dissimilarity and Non-metric multidimensional scaling (NMDS) to test for effects of site, region, species, and disease status. To test effects on microbiome stability (the ability of the organisms to maintain ideal skin communities), we will calculate the multidimensional area of minimum convex polygons fit to clusters of species using 'ordiareatest' function from the 'vegan' package.The PI along with assistance from the two host scientists will modify and improve the Disease Ecology section of the Ecosystem Science and Management Course (AGSC 5510/7510) at TSU. Specifically, we will modify this portion of the course to include lecture material on analysis of wildlife and zoonotic disease prevalence along with analysis of 16s DNA sequencing datasets. in RStudio. Additionally, will use Analysis of Similarity (ANOSIM) to examine differences in 16s microbial communities among ticks that tested positive for focal pathogens and among ticks collected from the vertebrate host groups and from drag samples.The PI and host scientists will meet to discuss potential plans for future research collaborations periodically throughout the funding period. In addition to research collaborations, the project participants will meet to discuss the potential of developing additional lecture material on wildlife and zoonotic disease detection and monitoring.

Progress 06/01/20 to 12/31/23

Outputs
Target Audience:The target audience for this research includes the United States Forest Service, the employees of the Bankhead National Forest, and graduate students enrolled in the AGSC 5510/7510 Ecosystem Science and Management course, along with undergraduate students enrolled in the AGSC 2400 Fundamentals of Environmental Science cours. In addition, the target audience includes any individuals that utilize public spaces, forests, and other natural areas for recreation. Outreach efforts included in-classroom lectures of microbiome research and disease ecology, which was incoporated in to the AGSC 5510/7510 course curriculum. In addition, partial study results were communicated to the general public and the United States Forest Service during a PhD disseration defense. Further planned dissemination will include an oral presentation and a peer-reviewed manuscript that will be submitted in December 2024. Changes/Problems:The only major change that we made during this project was a change in the sequencing approach. We were able to access a much deeper sequencing reading approach, which deviated from our initial experimental plan. We are excited about the results that we obtained and we will be sure to notify NIFA once results have been published. What opportunities for training and professional development has the project provided?The PI, Dr. William Sutton has been able to gain considerable lab-based experience, primarily in the areas of DNA extraction and qPCR analysis. The microbiome analysis has provided the PI with an additional set of training, particularly in the fields of bioinformatics and microbiome data analysis. The PI will now continue analysis on the lab results to publish a peer-reviewed manuscript that will document the experimental findings. Lastly, incorporation of additional teaching materials has greatly improved the AGSC 550/7500 course, which will be taught again in fall 2025. How have the results been disseminated to communities of interest?As explained above, we have incorporated the classroom lecture materials and have disseminated partial results to he US Forest Service. In addition, partial results have been disseminated in one dissertation and will be presented via a peer-reviewed manuscript, which will be submitted for potential publication in fall 2024/winter 2025. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have established collaborations with MTSU and the Tennessee Department of Health during this research period. Although COVID 19 initially limited research participation among the research groups, we have been able to re-establish collaborations and have been able to extract DNA samples from tick specimens and have completed all 16S microbiome sequencing. We have incorporated a microbiome section in the AGSC 5510/7510 course which was instructed inFall 2023at Tennessee State University. The Disease Ecology and Ecosystem One Health lectures have been completed and were incorporated in the AGSC 5500/7500 course in fall 2023. For objective 3, we have already had group discussions and will likely submit an additional proposal through NIFA during the 2024/2025 window.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Wildlife and Tick Responses to Forest Management: Prevalence of Tick-Borne Pathogens and Implications for One Health.


Progress 06/01/22 to 05/31/23

Outputs
Target Audience:The target audience for this research includes the United States Forest Service, the employees of the Bankhead National Forest, and graduate students enrolled in the AGSC 5510/7510 Ecosystem Science and Management course, along with undergraduate students enrolled in the AGSC 2400 Fundamentals of Environmental Science cours. In addition, the target audience includes any individuals that utilize public spaces, forests, and other natural areas for recreation. Outreach efforts included in-classroom lectures of microbiome research and disease ecology, which was incoporated in to the AGSC 5510/7510 course curriculum. In addition, partial study results were communicated to the general public and the United States Forest Service during a PhD disseration defense. Further planned dissemination will include published manuscripts that are currently being drafted and will be submitted in spring/summer 2023. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PI, Dr. William Sutton has been able to gain considerable lab-based experience, primarily in the areas of DNA extraction and qPCR analysis. The microbiome analysis has provided the PI with an additional set of training, particularly in the fields of bioinformatics and microbiome data analysis. Incorporation of additional teaching materials has greatly improved the AGSC 550/7500 course. How have the results been disseminated to communities of interest?As explained above, we have incorporated the classroom lecture materials and have disseminated partial results to he US Forest Service. In addition, partial results have been disseminated in one dissertation and will be presented via a peer reviewed manuscript. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete at microbiome sequencing by the completion of this reporting period.

Impacts
What was accomplished under these goals? We have established collaborations with MTSU and the Tennessee Department of Health during this research period. Although COVID 19 initially limited research participation among the research groups, we have been able to re-establish collaborations and have been able to extract DNA samples from tick specimens and have conducted pathogen testing. We are currently scheduled to ship additional samples for microbiome sequencing in Fall 2023. We have incorporated a microbiome section in the AGSC 5510/7510 course which was instructed in Fall 2023 at Tennessee State University. The Disease Ecology and Ecosystem One Health lectures have been completed and were incorporated in the AGSC 5500/7500 course in fall 2023. For objective 3, we have already had group discussions and are planning submission of additional proposals.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Wildlife and Tick Responses to Forest Management: Prevalence of Tick-Borne Pathogens and Implications for One Health.


Progress 06/01/21 to 05/31/22

Outputs
Target Audience:The target audience for this research includes the United States Forest Service, the employees of the Bankhead National Forest, and graduate students enrolled in the AGSC 5510/7510 Ecosystem Science and Management course, along with undergraduate students enrolled in the AGSC 2400 Fundamentals of Environmental Science cours. In addition, the target audience includes any individuals that utilize public spaces, forests, and other natural areas for recreation. Outreach efforts included in-classroom lectures of microbiome research and disease ecology, which was incoporated in to the AGSC 5510/7510 course curriculum. In addition, partial study results were communicated to the general public and the United States Forest Service during a PhD disseration defense. Further planned dissemination will include published manuscripts that are currently being drafted and will be submitted in spring/summer 2023. Changes/Problems:This project was stalled greatly by COVID-19 travel restrictions. However, we aim to be fully back on track with completion of original project goals. What opportunities for training and professional development has the project provided?The PI, Dr. William Sutton has been able to gain considerable lab-based experience, primarily in the areas of DNA extractionand qPCR analysis. The microbiome analysishas providedthe PI with an additional set of training, particularly in the fields of bioinformatics and microbiome data analysis. Incorporation of additional teaching materials has greatly improved the AGSC 550/7500 course. How have the results been disseminated to communities of interest?As explained above, we have incorporated the classroom lecture materials and have disseminated partial results to he US Forest Service. In addition, partial results have been disseminated in one dissertation and will be presented via a peer-reviewed manuscript. What do you plan to do during the next reporting period to accomplish the goals?We plan on sequencing additional microbiome samples for the remaining tick samples included in the study. We aim to complete this primarily in spring 2022. Results from this analysis will be incorporated in additional lectures and will also be prepared for peer-review article submission.

Impacts
What was accomplished under these goals? We have established collaborations with MTSU and the Tennessee Department of Health during this research period. Although COVID 19 initially limited research participation among the research groups, we have been able to re-establish collaborations and have been able to extract DNA samples from tick specimens and have conducted pathogen testing. We arecurrently scheduled to ship additional samples for microbiome sequencingin Spring 2023. We have incorporated a microbiome section in the AGSC 5510/7510 course which is currently being instructed (Fall 2021) at Tennessee State University. The Disease Ecology and Ecosystem One Health lectures have been completed and were incorporated in the AGSC 5500/7500 course in fall 2021. For objective 3, we have already had group discussions and are planning submission of additional proposals in fall 2023.

Publications

  • Type: Theses/Dissertations Status: Accepted Year Published: 2022 Citation: Wildlife and Tick Responses to Forest Management: Prevalence of Tick-Borne Pathogens and Implications for One Health.


Progress 06/01/20 to 05/31/21

Outputs
Target Audience:The target audience for this research includes the United States Forest Service, the employees of the Bankhead National Forest, and graduate students enrolled in the AGSC 5510/7510 Ecosystem Science and Management course. In addition, the target audience includes any individuals that utilize public spaces, forests, and other natural areas for recreation. Outreach efforts included in-classroom lectures of microbiome research and disease ecology, which was incoporated in to the the AGSC 5510/7510 course curriculum.In addition, partial study results were communicated to the general public and the United States Forest Service during a PhD disseration defense and will be inlcuded in a doctoral dissertation. Further planned dissemination will include published manuscripts. Changes/Problems:COVID 19 presented major problems with access to lab space. However, as a No-Cost extension was granted, we see no issues with achieiving project objectives by the project end date of May 2022. What opportunities for training and professional development has the project provided?The PI, Dr. William Sutton has been able to gain considerable lab-based experience, primarily in the areas of DNA extraction and qPCR analysis. The microbiome analysis (scheduled for fall 2021) will provide the PI with an additional set of training, particularly in the fields of bioinformatics and microbiome data analysis. How have the results been disseminated to communities of interest?Partially. As explained above, we have incorporated the classroom lecture materials and have disseminated partial results to the US Forest Service. Our final study results will be disseminated via peer-reviewed publication. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete the microbiome analysis in fall 2021 and will likely complete the bioinformatics analysis in spring 2022. This will involve travel to MTSU to complete the laboratory analysis and library prepfor this portion of the work.We will hold bi-montlhy progress calls via zoom to discuss future objectives and to assure that project objectives are completed in a timely manner.

Impacts
What was accomplished under these goals? We have established collaborations with MTSU and the Tennessee Department of Health during this research period. Although COVID 19 initially limited research participation among the research groups, we have been able to re-establish collaborations and have been able to extract DNA samples from tick specimens and have conducted pathogen testing. We are planning to complete microbiome analysis in late fall 2021 at MTSU. We have incorporated a microbiome section in the AGSC 5510/7510 course which is currently being instructed (Fall 2021) at Tennessee State University. The Disease Ecology and Ecosystem One Health lectures will include Microbiome components for the course. Objective 3 will be handled during spring 2022 after the group has completed a majority of the molecular analyses. We will discuss future research opportunities that might become available based on the results gained from the current project.

Publications