Progress 03/15/24 to 03/14/25
Outputs
Target Audience:Researchers, Animal Scientists, Veterinary Students, Veterinarians, Breeders Changes/Problems:Our initial plan was to use the NanoString platform in combination with ACD Biotechne nucleic acid probes for spatial sequencing at the University of California, Irvine. This approach was selected due to its ability to localize gene expression with high specificity using targeted in situ hybridization. However, upon conducting preliminary trials, we encountered significant technical challenges that impacted data quality. We observed that the sequencing output lacked the expected resolution and sensitivity required to accurately map CHIR expression across different intestinal cell populations. This limitation was particularly concerning given the complexity of immune cell interactions within the jejunal tissue. A major factor contributing to the poor sequencing quality was the repeated heating steps involved in the NanoString protocol. These heating cycles were necessary for sample preparation but likely led to RNA degradation, reducing the reliability of gene expression signals. Given that RNA integrity is crucial for spatial transcriptomic accuracy, we determined that an alternative approach was necessary. To overcome these challenges, we transitioned to the 10x Visium platform at the University of California, Los Angeles. This decision was made after evaluating its advantages in providing higher-quality spatial transcriptomic data while avoiding the pitfalls of the NanoString methodology. Unlike the NanoString protocol, the 10x Visium workflow does not require extensive heating steps. This reduces RNA degradation, ensuring that gene expression data is preserved and more accurately reflects the spatial distribution of immune-related genes, including CHIR-B2, SHP-1, and SHP-2. One of the key limitations of the NanoString approach was that it required pre-selection of regions of interest (ROIs) for sequencing. This meant that only predefined tissue sections could be analyzed, potentially leading to missed spatial relationships in gene expression outside the selected ROIs. In contrast, the 10x Visium platform sequences the entire tissue section, allowing us to capture a broader, unbiased view of transcriptomic changes across different intestinal regions. This is particularly advantageous for studying complex tissue structures like the jejunum, where immune responses may vary across different cellular niches. The 10x Visium platform also offers higher spatial resolution and the ability to integrate seamlessly with single-cell RNA sequencing data, providing a more comprehensive understanding of cell type-specific immune responses to coccidiosis infection. By switching to the 10x Visium platform at UCLA, we have significantly improved the reliability and resolution of our spatial transcriptomic data. This change ensures that our findings on CHIR localization, immune signaling, and disease susceptibility are supported by robust and high-quality sequencing data. Additionally, the whole-tissue sequencing capability of the 10x platform allows us to explore unexpected spatial gene expression patterns that may have been overlooked using a targeted ROI-based approach. This adjustment represents a critical improvement in our experimental design, ensuring that we generate the most accurate and informative data possible for understanding the role of CHIR-B2, SHP-1, and SHP-2 in the intestinal immune response to coccidiosis.? What opportunities for training and professional development has the project provided?This project has provided significant training and professional development opportunities, particularly for postdoctoral researcher Dr. Brandi Sparling, who recently graduated with a PhD specializing in sequencing data analysis. She played a crucial role in performing all data analysis work, including preprocessing, quality control, and interpretation of both scRNA-seq and spatial transcriptomic datasets. Through this experience, she has expanded her expertise in bioinformatics, including advanced computational methods for single-cell analysis, spatial mapping, and statistical correlation techniques. Additionally, she has gained valuable hands-on experience in integrating multi-omic data, applying machine learning algorithms for cell type identification, and developing robust workflows for differential gene expression analysis. Her contributions to this project have positioned her well for future academic and industry roles that require expertise in high-throughput sequencing technologies and bioinformatics. Beyond technical skills, she has been actively involved in manuscript preparation and data visualization, enhancing her ability to communicate complex biological insights effectively. Her role in this project will significantly contribute to her professional growth and career trajectory in computational biology and transcriptomics. This project has provided valuable training and professional development opportunities for Jeselle-Ann Laxa, a DVM 2027 veterinary student, as part of the Veterinary Scholar program at WU CVM, to be expanded in the upcoming2025 Veterinary Summer Research Program. She will play a key role in conducting histopathological analyses using hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) staining to validate the inflammatory status of intestinal tissues collected in this study. Through this work, she will receive hands-on laboratory training in histological techniques, including tissue sectioning, staining protocols, and microscopy analysis. She will gain experience in recognizing and interpreting histopathological changes associated with coccidiosis, such as inflammatory cell infiltration, epithelial damage, and lesion severity scoring. Additionally, she will contribute to validating molecular findings from spatial transcriptomics and single-cell RNA sequencing, bridging histological and molecular data to provide a more comprehensive understanding of disease pathology. Beyond laboratory techniques, she will develop critical skills in data analysis and scientific interpretation. She will be trained in quantifying histological features, performing statistical analyses, and correlating histopathological findings with molecular and clinical data. This experience will enhance her ability to analyze research data and contribute meaningfully to scientific discussions. To further her professional development to be a veterinary pathologist, she will present her findings at a scientific conference, with the Poultry Science Association (PSA) and the American Association of Avian Pathologists (AAAP) as potential venues. These conferences will provide her with opportunities to engage with experts in veterinary pathology, poultry health, and immunology, as well as network with researchers and industry professionals. The experience of preparing and delivering a scientific presentation will strengthen her communication skills and prepare her for future roles in research and veterinary medicine. Overall, this project will provide her with a well-rounded research experience, equipping her with essential laboratory skills, analytical expertise, and scientific communication abilities that will support her future career as a veterinarian with a strong foundation in research. As a co-Principal Investigator and new faculty, this project has provided significant professional development opportunities for Dr. Theros Ng by allowing for the training and mentorship of both postdoctoral fellows and veterinary scientists. Through this study, he has played a critical role in guiding postdoctoral researcher Dr. Brandi Sparling in advanced sequencing data analysis and supporting veterinary student Jeselle-Ann Laxa in histopathological and immunohistochemical techniques. This mentorship has further strengthened his expertise in research training and academic leadership, essential for fostering the next generation of scientists and veterinary professionals in the field of poultry immunology and pathology. Additionally, this project has enabled him to deepen expertise in integrating multi-omic data, spatial transcriptomics, and immunopathology in poultry disease research. The findings from this study provide new insights into the molecular mechanisms underlying coccidiosis susceptibility and resistance in MHC-B haplotype chickens, which will contribute to his broader research portfolio in avian immunogenetics. As part of the dissemination of this research, he will present the findings at the Poultry Science Association (PSA) annual meeting and at NE2334: Genetic Bases for Resistance and Immunity to Avian Diseases, a multi-institutional research group focused on understanding genetic and immunological mechanisms of disease resistance in poultry. Presenting at these conferences will provide opportunities to engage with leading researchers, foster collaborations, and contribute to discussions on improving poultry health through genetic and immunological advancements. This project has not only expanded his research capabilities but has also strengthened professional networks within the poultry research and veterinary communities. By mentoring trainees and disseminating findings at national and international conferences, he continues to contribute to the advancement of avian disease research while building a strong foundation for future collaborative projects in poultry immunogenetics. How have the results been disseminated to communities of interest?The results of this study have been actively disseminated to scientific communities and stakeholders through multiple presentations at regional, national, and institutional conferences. These efforts have provided opportunities to share our findings with experts in poultry immunogenetics, veterinary medicine, and avian disease research, fostering discussions and collaborations. These presentations have ensured that our research findings reach relevant academic, industry, and veterinary communities, promoting awareness of the genetic and molecular factors influencing coccidiosis resistance. Future dissemination efforts will include submission of manuscripts to peer-reviewed journals and continued engagement with stakeholders through additional conference presentations and collaborative discussions. What do you plan to do during the next reporting period to accomplish the goals?The upcoming phases of the project include the completion of spatial sequencing data analysis, followed by the commencement of scRNA-seq analysis. The next step is to complete the statistical analysis of different haplotype-infection status groups to identify the cells and genes implicated in the immunogenetic response. Additionally, integrating simultaneously generated single-cell sequencing data with phenotypic data (body weight changes and lesion scores) will allow us to correlate transcriptomic changes with organ-, tissue-, and whole-body-level responses. This will involve differential gene expression analysis, identification of specific immune cell subpopulations, and GO enrichment analysis to uncover the functional implications of CHIR-B2 signaling. Additionally, validation studies using immunohistochemistry and qRT-PCR will be performed to further confirm the expression patterns observed in sequencing data. Comparative analyses of immune cell composition between B19 and BQ chickens will be performed to determine whether specific cell types contribute to disease susceptibility. The identification of rare immune cell populations will provide new insights into the immunogenetic basis of coccidiosis resistance. Following the completion of analyses, the findings will be compiled for publication in a peer-reviewed journal.
Impacts
What was accomplished under these goals?
The objective of this study is to identify and characterize CHIR-B2 and its association with SHP-1 and SHP-2 in the chicken intestine in the context of coccidiosis. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics, we aim to localize CHIRs within different intestinal cell populations and correlate their expression with disease susceptibility in MHC-B haplotype chickens (B19 vs. BQ). This study seeks to provide a comprehensive understanding of how immune inhibitory CHIR-B types contribute to susceptibility to coccidiosis and how these findings can be leveraged to develop breeding strategies for disease-resistant poultry. Two animal studies have been successfully completed. Homozygous B19 and BQ chickens were obtained from Dr. Robert L. Taylor at West Virginia University, confirmed through LEI0258 PCR, and allocated to respective treatment groups of control and infected. The infected groups were housed in a USDA BSL-2 certified facility to prevent cross-contamination. Coccidiosis challenge was administered on day 4 post-hatch via oral gavage with a 10x dose of COCCIVAC-D2 vaccine, a live non-attenuated blend of multiple Eimeria species. Weekly performance data, including bird weight, feed intake, and behavioral observations, were meticulously recorded to monitor the impact of infection on growth and development. Necropsy was performed on day 21, and tissue samples were collected from the jejunum for histopathology, scRNA-seq, and spatial transcriptomic analysis. Intestinal lesion scoring, using a 0-4 scale, was conducted across the jejunum, ileum, and cecum. Results revealed a stark contrast between genetic lines, with B19 chickens showing significantly higher lesion scores, indicative of severe infection and intestinal damage. Preliminary findings indicate significantly higher lesions in B19 chickens, correlating with their more severe disease phenotype. Following tissue collection, single-cell dissociation was optimized using the Multi Tissue Dissociation Kit and gentleMACS Octo Dissociator. Viability was assessed using 7-AAD staining, ensuring the removal of dead cells before sequencing. Cell sorting was performed via flow cytometry, and high-quality live cells were processed for library preparation using the 10x Genomics Chromium platform at UCLA. Sequencing was performed on the Illumina NovaSeq X plus system at 25B, generating high-depth transcriptomic data across all experimental groups. Initial quality control assessments, performed using FASTQC and MultiQC, confirmed high quality and sufficient sequencing depth. Sequence barcodes and unique molecular identifiers (UMIs) were assigned, followed by read alignment to the latest chicken reference genome (GCA_016700215.2). Downstream analysis, including normalization, clustering, and dimensionality reduction (UMAP and t-SNE), will commence once the spatial sequencing data is fully integrated. For spatial transcriptomics, flash-frozen jejunum tissues were sectioned and stained for histopathological evaluation. The 10x Genomics Visum spatial sequencing tissue optimization kit was used to optimize cDNA reagent permeation. cDNA libraries were constructed from the sectioned samples on the spatial sequencing slides. Quality control was performed using NanoDrop and TapeStation. Sequencing was performed on the NovaSeq X Plus 10B for 100 cycles. The stained H&E-stained brightfield images were imported into QuPath (v0.5.1) or Aperio ImageScope (v12.3.3) for extraneous image area outside of the fiducial border. For quality export and downstream visualization, only QuPath images rendered in RGB were exported. For read alignment and gene expression quantification we used Space Ranger(v3.1.2) count function which took raw fastq files, aligned the reads to a custom broiler reference, and mapped the expressions of genes spatially using the new QuPath tissue images. A custom broiler reference index based off of GCF_016699485.2_bGalGal1.mat.broiler.GRCg7b was made using the spaceranger mkgtf function. Quality control was then checked by the resulting interactive web_summary reports and the .cloupe files were visually checked in Loupe Browser. For groupwise analysis, the spaceranger aggr function, and the previous step's spatial folder, cloupe file, and molecule_h5 file was used to create a single feature-barcode matrix. A metadata .csv file was created and appended to the obs attribute of the H5 AnnData object to annotate the haplotype-infection status of our strip-plot experimental design. To enhance clustering accuracy while preserving spatial relationships, a spatial nearest-neighbor graph was constructed using STAGATE (v02.12.25). Unlike batch correction methods like Harmony or BBKNN, STAGATE maintains tissue architecture and spatial gene expression continuity while reducing noise. The spatially aware embeddings generated by STAGATE were then used to perform Leiden clustering and UMAP visualization of clusters. To assign putative cell identities to these spatial clusters, a custom automatic annotation approach was employed. First, SingleR (v2.4.1) was applied to compare spatially resolved transcriptomes against ours and others' previous bulk RNA-seq reference datasets (B cells, macrophages, monocytes, T cells, M cells, IFE cells, and fibroblasts) (Overby et al., 2021, Zeinali et al., 2024, and de Vos et al., 2023), assigning the most likely cell type based on resampling-based (n_bootstrap = 1000) empirical p-values, and a 95% confidence threshold. However, additional refinement was performed using TabulaSapiens_v2, a comprehensive single-cell transcriptomic atlas that maps over 1.1 million cells from 28 organs across 24 normal human subjects (https://tabula-sapiens.sf.czbiohub.org/) (Tabula Sapiens Consortium, 2022). Shared genes between the broiler and human datasets were identified, and K-Nearest Neighbors (KNN) classification approach (k=5) was trained to match spatial clusters to their most similar cell types based on cosine similarity (considers relative expression levels of all shared genes). These labels were applied back to our Leiden clusters and UMAP visualizations, as well as each individual samples' H&E images for morphological assessment. We are currently correlating these spatial expression profiles with lesion severity and other histological features. Early results indicate that CHIR-B2 expression is highly upregulated in specific immune cell populations within the lamina propria of B19 chickens, whereas BQ chickens exhibit more localized SHP-1 expression at the base of intestinal crypts, supporting its role in epithelial proliferation and immune modulation. Further analyses are ongoing to determine the statistical significance of these observations and their implications for immune response differences between the two genetic lines. The integration of lesion scores with spatial transcriptomic data is underway. Statistical analyses, including Spearman correlation and principal component analysis (PCA), are being conducted to assess the relationship between intestinal damage severity and CHIR-B2, SHP-1, and SHP-2 expression patterns. The project has made significant progress, with all animal experiments completed and sequencing efforts largely finalized. The integration of spatial transcriptomic data with clinical lesion scores is revealing key insights into the role of genetics in coccidiosis susceptibility. The upcoming single-cell analysis phase will further elucidate immune cell interactions and provide a comprehensive view of immune response variation between genetic lines. Our findings have strong implications for poultry breeding strategies and disease resistance mechanisms, aligning well with the USDA NIFA's priority to enhance animal health and disease resistance.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
NE2334 Annual Meeting: Genetic Basis for the Resistance and Immunity to Avian Diseases, Western University of Health Sciences College of Veterinary Medicine Station Report. Champaign, IL, October 1st to 2nd.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Poultry Science Association Annual Meeting 2024, Louisville, KY. July 15th to July 18th, 2024: Targeted RNA in situ hybridization to determine the spatial expression of the Cluster Homolog of Immunoglobulin-like Receptors (CHIR) in the chicken intestine after coccidia vaccine challenge (oral presentation)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Research Day, College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA. March 25th, 2024
Title: Targeted RNA in situ hybridization to determine the spatial expression of the Cluster Homolog of Immunoglobulin-like Receptors (CHIR) in the chicken intestine after coccidia vaccine challenge (poster presentation)
Authors: Hoangvi Le*, Brandi Sparling, Yvonne Drechsler, Theros T. Ng
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Progress 03/15/23 to 03/14/24
Outputs
Target Audience:Researchers, animal science and veterinary students Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The postdoctoral fellow, Dr. Theros T. Ng, has been hired as an Assistant Professor in Foundational Sciences in the College of Veterinary Medicine at Western University of Health Sciences, Pomona, CA partly due to his work on this project as well as other USDA funded projects. Dr. Ng has been requested to be added as co-PD of this study and working diligently to complete this project with his Research Associate, Hoangvi Le, recently hired in the same college immediately after graduating with a Master's degree and is receiving training in bioinformatics, and cutting edge sequencing techniques. Postdoctoral fellow Brandi Sparling has provided support with the experimental phase and will be analyzing data bioinformatically, learning about diverse sequencing analyses. How have the results been disseminated to communities of interest?Due to limited results before sequencing the samples, this project has been introduceds and input solicited at the annual NE1834 meeting in 2023. What do you plan to do during the next reporting period to accomplish the goals?A selected portion of the cryopreserved jejunum tissue, representative of the critical site of coccidial infection and pathology, will undergo processing to ensure preservation of spatial information prior to sequencing. This process will be processed using the 10x Visium Spatial Sequencing platform at the University of California, Irvine (UCI). Subsequently, the generated spatial sequencing data will be analyzed, with a particular focus on optimizing the analysis pipeline to elucidate the spatial distribution and differential expression patterns of key genes of interest, including CHIR-B, SHP-1, and SHP-2. We have ordered the reagents and are in the process to schedule with UC Irvine. Concurrently, the single-cell homogenate derived from the harvested tissue will undergo library preparation using the 10x single-cell 3' gene expression kit, followed by sequencing at a depth of 50 million read depth, also conducted at UCI. This comprehensive approach will enable the robust characterization of gene expression profiles within individual cells, facilitating a deeper understanding of cellular heterogeneity and the molecular mechanisms underlying host response to coccidial infection. Upon completion of single-cell sequencing, efforts will be directed towards the design and synthesis of nucleotide probes specifically targeting the CHIR-B, SHP-1, and SHP-2 genes. These probes will serve as invaluable tools for nucleotide staining of histology slides prepared from formalin-preserved tissues, enabling precise visualization of gene expression patterns within the tissue microenvironment. High-resolution imaging of the stained slides will be conducted using fluorescent microscopy techniques, further enhancing our ability to discern spatial relationships and expression dynamics of the target genes. The culmination of these analyses and experimental procedures will yield a comprehensive dataset elucidating the differential expression profiles of CHIR-B, SHP-1, and SHP-2 genes in the context of coccidiosis susceptibility. Specifically, comparisons will be drawn between the BQ line, known for its coccidiosis resistance, and the B19 line, characterized by susceptibility to coccidial infection, shedding light on potential molecular determinants underlying differential disease susceptibility. The dissemination of findings derived from this comprehensive investigation will be presented at the 2024 Poultry Science Association Annual Meeting in Louisville, Kentucky. The responsibility of presenting the data will be entrusted to the dedicated Research Associate, Hoangvi Le, whose commitment and contributions have been integral to the success of the study. Following presentation at the conference, efforts will be directed towards manuscript preparation, with the aim of providing a detailed account of the study findings for dissemination within the scientific community, thereby advancing our understanding of host-pathogen interactions in poultry health and informing future research endeavors in the field.
Impacts
What was accomplished under these goals?
Fertile eggs of specific genotypes, namely BQ/BQ and B19/B19, were procured from Dr. Robert L. Taylor Jr. at West Virginia University. Upon receipt, these eggs underwent safe handling procedures to maintain their viability during transit to our research facility. Upon arrival, the eggs were promptly incubated under controlled conditions until hatching, following which the newly hatched chicks were individually identified via wing-tagging and transferred to a designated brooder cage within our vivarium facility. A nutritionally balanced corn-based meshed chick feed was providedad libitumto ensure optimal growth and development, accompanied by unrestricted access to water. To ascertain the genetic sex of the chicks, a minimally invasive blood sampling procedure was performed on day 2 post-hatch, with one droplet of blood being collected from each chick. Subsequently, DNA extraction was carried out utilizing Chelex beads, a well-established method known for its efficacy in isolating high-quality genetic material from biological samples. Polymerase Chain Reaction (PCR) was then employed, following optimization procedures, to determine the sex of the chicks utilizing CHD-1 F/R primers. Upon sex determination, male chicks were adopted out. Female chicks of BQ and B19 genotypes, were randomly allocated to treatment groups, resulting in a 2x2 factorial design comprising control and infected cohorts. Weekly weight monitoring of the chicks facilitated the assessment of growth patterns and potential alterations induced by experimental conditions. Control groups, consisting of BQ (n = 10) and B19 (n = 9) lineages, were maintained separately from their infected counterparts. Infected groups, comprising BQ (n = 11) and B19 (n = 10) lineages, were housed in dedicated isolators within a controlled environment to prevent cross-contamination. At day 4 post-hatch, chicks assigned to the infected treatment groups were subjected to oral gavage with a 10x dose of a commercial coccidia vaccine (COCCIVAC-D2, Merck, Kenilworth, NJ), mimicking a challenge scenario. Subsequently, at day 21 post-hatch, chicks from each treatment group (n = 5) were euthanized for comprehensive evaluation. Upon euthanasia, the jejunum, a key site of coccidial infection and pathology, was harvested. Tissue samples were processed utilizing various methodologies to facilitate downstream analyses. Cryopreservation of tissue sections was performed using specialized techniques to maintain cellular integrity, followed by storage at -80ºC to ensure long-term preservation. Simultaneously, another portion of the harvested tissue underwent homogenization in sterile phosphate-buffered saline (PBS) to generate a single-cell suspension using the Miltenyi gentleMACs homogenizer. Following filtration through a sterile 40 μm cell strainer and subsequent washing steps, the cells were suspended in cryopreservation media and gradually frozen using a controlled freezing apparatus. These cryopreserved cell suspensions were stored at -80ºC pending further analysis. Additionally, tissue sections were fixed in 10% Neutral Buffered Formalin for histological evaluation and nucleic acid-based probe staining in the future. Preliminary analysis of the collected data revealed distinct differences in lesion scores between B19-infected and BQ-infected chickens, consistent with expectations based on prior literature. While both infected groups exhibited weight loss following infection, statistical significance was not attained, likely attributed to the relatively modest sample size inherent to this genomic study. To augment our understanding of the molecular mechanisms underlying the observed phenotypic responses, cryopreserved tissue samples have been dispatched to the Genomic Research and Technology Hub at the University of California Irvine. Utilizing the spatial sequencing technology offered by the 10x Genomics Visium platform, comprehensive spatial gene expression profiles will be generated, elucidating spatially resolved transcriptomic signatures within the intestinal tissue microenvironment. Concurrently, the cryopreserved cell homogenate has been forwarded to the same core laboratory at UC Irvine, where live cell sorting employing 2-AAD dye via flow cytometry will be conducted to isolate viable cell populations of interest. Subsequent single-cell RNA sequencing utilizing the 10x Genomics Single-cell 3' Gene Expression Kit at a depth of 50 million reads depth will enable in-depth characterization of cellular heterogeneity and gene expression dynamics underlying the host response to coccidial infection. This comprehensive approach, combining traditional histopathological evaluation with cutting-edge genomic analyses, promises to unravel intricate molecular mechanisms governing host-pathogen interactions in the context of coccidiosis, thereby facilitating the development of novel therapeutic interventions and management strategies for this economically significant poultry disease.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
NE1834 meeting 2023: Drechsler Yvonne, Ng Theros, Sparling Brandi: WU Station report
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