Progress 07/01/24 to 06/30/25

Outputs
Target Audience:During the current reporting period, the project primarily targeted graduate-level students in animal and human health sciences fields by providing them with practical experiences in laboratory instruction, molecular biology, and immunological research methods. One graduate student (Tanner Scull) participated in hypothesis-driven research activities aimed at understanding immune dysfunction in bovine leukemia virus (BLV)-infected dairy cows. His involvement included PBMC isolation, flow cytometry, transcriptomic analysis, and engagement in data interpretation and experimental design. These experiences are essential for developing the next generation of scientists equipped to address infectious diseases affecting livestock health and productivity. This audience was specifically targeted to align with the project's mid-term goal of supporting scientific training and workforce development in veterinary immunology, large animal research, and bioinformatics. By embedding student researchers into active investigative roles, this project fosters technical proficiency, critical thinking, and data literacy, which are directly applicable to careers in veterinary and agricultural sciences. In addition to graduate student training, institutional academic and administrative personnel were engaged during this reporting period through routine project coordination, research compliance, and data management. These include efforts related to IACUC protocol submissions, sample logistics, and fiscal management, which support the research infrastructure required to meet the project's objectives. Although no direct engagement with producers, veterinarians, or extension agents took place during this phase, the foundational data generated this year will inform future outreach efforts to dairy health professionals and livestock producers. These groups remain key long-term audiences for the project as it progresses toward generating applied knowledge and management strategies for controlling BLV-related immune dysfunction. Changes/Problems:During this reporting period, no significant changes were made to the overall goals, objectives, or scope of the project. However, we encountered a few minor but noteworthy adjustments in scheduling and methodology, described below. Single-Cell Data Timeline Adjustment The original timeline proposed acquiring and analyzing single-cell RNA-sequencing (scRNA-seq) data within the first project year. While we successfully screened approximately 400 animals for BLV proviral load and submitted samples for sequencing, the data delivery from the core facility was delayed and is now expected by the end of August 2025. This delay does not affect the scope of work, and we have used the interim time to optimize downstream protocols for data analysis and validation. Optimization of B and T Cell Sorting Protocols The original proposal anticipated B-cell sorting to begin earlier. However, method refinement took longer than expected due to variability in marker expression and optimization of staining conditions in bovine samples. Despite this, we have now established a reliable flow cytometry panel and successfully performed preliminary sorting. These delays were necessary to ensure reproducibility and quality for downstream transcriptomic and functional assays. In Vitro Co-Culture System Development The development of functional co-culture systems (Objective 3) was initially planned for later in the project timeline. However, we began pilot experiments earlier than anticipated to take advantage of protocol optimization downtime. This resulted in an internal timeline adjustment, but no change to scope or milestones. No Changes to Data Management Plan There were no modifications to the approved Data Management Plan. Data produced during this period (e.g., flow cytometry, qPCR results) are being securely stored and organized for future public sharing alongside scRNA-seq datasets once processing is complete. No Issues with Animal Protocols or Biohazards All animal handling and sampling procedures were conducted by approved IACUC protocols. No changes or problems involving animal welfare, biosafety, or compliance were encountered. Summary Although minor scheduling adjustments were made, particularly with single-cell sequencing and immune cell sorting workflows, these changes were managed without impacting the core goals or budget. The project remains on track for full completion, and adjustments made during this reporting period are expected to improve data quality and experimental reproducibility. What opportunities for training and professional development has the project provided?During this reporting period, the project provided intensive one-on-one mentoring and training for one graduate student (Tanner Scull). The student received hands-on instruction in multiple advanced laboratory techniques, including: - Peripheral blood mononuclear cell (PBMC) isolation from bovine whole blood; - Flow cytometry and fluorescence-activated cell sorting (FACS) for immune profiling; - RNA extraction and quantitative PCR for BLV detection; - Development of antibody panels specific for B and T cell markers; - Protocol optimization for downstream single-cell transcriptomics; - Early-stage co-culture modeling of immune cell interactions. Additionally, Tanner participated in project design discussions, contributed to protocol troubleshooting, and was involved in scientific writing and data preparation for presentations. These experiences strengthened his technical and critical thinking skills, positioning him for a future career in animal health and immunological research. The project also supported professional development through: - Participation in internal lab meetings; - Informal seminars and graduate-level discussions on host-pathogen interactions; - Networking opportunities with faculty from multiple departments at Texas Tech University. How have the results been disseminated to communities of interest?During the reporting period, preliminary results and methodological developments were shared through the following avenues: - Departmental presentations: The graduate student presented findings and methods at a departmental seminar in the Department of Veterinary Sciences at Texas Tech University. - Informal instruction: Findings and protocols were discussed during small-group sessions in graduate-level coursework, including classes focused on veterinary immunology and large animal internal medicine. - Research discussions: Key insights and workflow improvements were shared with collaborators at the Center for Biotechnology & Genomics, fostering awareness and potential cross-project integration. While broader outreach to stakeholders such as producers and veterinarians will be prioritized in future phases, this year's focus on technical development and training laid the groundwork for future knowledge transfer and stakeholder engagement. What do you plan to do during the next reporting period to accomplish the goals?In the upcoming reporting period, we plan to focus on the following key activities for each objective: - Objective 1: - Analyze the single-cell RNA sequencing data from selected BLV+ and BLV- animals. - Identify transcriptional profiles associated with disease status and immune cell subsets. - Generate preliminary figures and analyses to support upcoming conference abstracts and manuscripts. - Objective 2: - Perform B and T cell sorting on fresh samples using optimized panels. - Begin transcriptional profiling of sorted B cells using bulk RNA-seq or targeted qPCR. - Explore correlations between surface markers and BLV load or transcriptional state. - Objective 3: - Finalize protocols for immune cell co-culture and stimulation assays. - Run pilot experiments testing immune responsiveness in sorted B cells. - Collect phenotypic data (e.g., proliferation, cytokine expression) to determine functional differences between BLV+ and BLV- cells. Additionally, we plan to expand outreach activities, submit at least one abstract for a national conference, and begin manuscript preparation based on data from Objectives 1 and 2.

Impacts
What was accomplished under these goals? Bovine leukemia virus (BLV) is a widespread retroviral infection affecting over 40% of U.S. dairy cows. It silently impairs the animal's immune system, especially B cells, which are crucial for fighting infections. Despite its prevalence, we still lack a clear understanding of how the virus changes the immune system over time. This project aims to fill that gap by using cutting-edge techniques, including single-cell RNA sequencing and functional immune assays, to uncover the cellular and molecular effects of BLV. This research benefits dairy farmers, veterinarians, scientists, and animal health professionals. A better understanding of how BLV affects cattle can lead to better management strategies, earlier detection, and targeted treatments. These outcomes will help improve animal welfare, productivity, and the economic sustainability of dairy farms. Objective 1: Define PBMC transcriptional changes associated with BLV proviral load - Major activities: We screened approximately 400 cows for BLV proviral load using quantitative PCR and ELISA. From these, using statistical analyses of data including days in milk, lactation number, age, and disease status to control for confounding variables, we selected cows representing both low and high proviral load groups for advanced analysis. - Data collected: We processed and submitted blood samples from selected cows for single-cell RNA sequencing (scRNA-seq). This work completes all experimental preparation for Aim 1. - Outcome: We created a well-curated sample biobank and completed the complex process of preparing immune cells for scRNA-seq. Sequencing data will arrive in August 2025. This achievement is a foundational milestone enabling future discovery of cell-level changes caused by BLV. Objective 2: Define B cell-specific transcriptional phenotypes in BLV+ and BLV- cows - Major activities: We optimized and validated antibody panels for identifying B and T cells in cow peripheral blood using flow cytometry. - Data collected: Preliminary sorting protocols were implemented, and viable B cell populations were isolated. - Outcome: We established reproducible, in-house protocols for immune cell sorting in dairy cattle. These tools will allow high-resolution study of B cell dysfunction and are transferable to other ruminant immunology applications. Objective 3: Determine the functional consequences of B cell dysfunction using co-culture systems - Major activities: Initiated development of in vitro co-culture systems to simulate immune cell interactions under BLV-infected conditions. - Data collected: Pilot experiments are underway, focusing on optimizing culture conditions and immune cell viability. - Outcome: Early results are shaping protocols that will allow us to study functional defects in immune response. This is a critical step toward understanding how BLV weakens immunity in infected animals. Key Measurable Outcomes - New research capacity: We established protocols for PBMC isolation, cell sorting, and scRNA-seq preparation in dairy cattle--a technical advancement for the field. - High-throughput animal screening: Successfully tested 400 cows for BLV, allowing precise selection for future in-depth analysis. - Student development: A graduate student was trained in molecular and immunological techniques, supporting future workforce development. - Institutional collaboration: Strengthened ties between the Department of Veterinary Sciences and the Center for Biotechnology & Genomics at Texas Tech University, promoting cross-disciplinary innovation. Impact Summary: This year, we took major steps to understand how a common virus affects the immune system in dairy cows. We tested hundreds of animals and sent samples for high-tech analysis that will tell us how the virus changes individual immune cells. We also developed lab methods to study these cells and how they interact. This work helps farmers and veterinarians by laying the groundwork for better ways to manage disease and improve herd health. Our efforts are already producing new tools, training future animal scientists, and building partnerships that will benefit livestock health and productivity.

Publications

• Type: Peer Reviewed Journal Articles Status: Accepted Year Published: 2024 Citation: Scull, T. F., Strieder-Barboza, C., & Benitez, O. J. (2024). Peripheral Blood Mononuclear Cell Transcriptome of Dairy Cows Naturally Infected with Bovine Leukemia Virus. *Pathogens*, 13(10), 885. https://doi.org/10.3390/pathogens13100885