Integrating clinical, data-driven and in-vitro approaches to the study of host-pathogen interactions in bovine digital dermatitis
Lead Research Organisation:
Royal Veterinary College
Department Name: Clinical Sciences and Services
Abstract
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Technical Summary
Genotypic, pedigree and phenotypic clinical data are already available for a population of 3,286 Holstein dairy animals. A new population of 500 dairy heifers will be prospectively monitored. We will be inspecting feet monthly and record presence of digital dermatitis (DD) lesions. DD resistance phenotypes and a novel DD progression phenotype will be developed. Blood samples for genotyping, whole genome sequencing and serum harvesting will be collected. Individual animal data will be analysed to determine genetic variants associated with the DD-related phenotypes. We will also derive genomic breeding values for each animal and DD phenotype. Foot skin biopsies will be performed on 24 animals genetically resistant to DD that will remain healthy throughout the study and 48 genetically susceptible animals (including recovered and chronically infected). Foot skin biopsy samples will be processed for total bulk RNA-sequencing in order to define DD resistance and progression transcriptomic signatures. We will perform single cell RNA-Seq in a subset of samples to dissect which cells are the key players driving DD resistance and progression. We will isolate fibroblasts and keratinocytes from cattle foot skin of the three respective biopsied groups and will co-incubate them with both a poly-treponemal mixture and an individual treponemes species. After co-incubation we will undertake RNA sequencing to determine global differential mRNA expression. The cattle foot skin culture model will be combined with a transepithelial cell migration assay to assess migration of granulocytes through bovine foot skin keratinocytes and fibroblasts. We will prioritise candidate genes and genetic variants for DD using a combined data-driven and biology driven approach, and will validate key findings using in vitro techniques. Outcomes from the previous steps will be integrated in a series of simulation studies to determine optimal biology-driven selective breeding strategies for DD control.
Publications
Anagnostopoulos A
(2024)
Association between a genetic index for digital dermatitis resistance and the presence of digital dermatitis, heel horn erosion, and interdigital hyperplasia in Holstein cows.
in Journal of dairy science
Tarsani E
(2025)
Genome-wide association studies of dairy cattle resistance to digital dermatitis recorded at four distinct lactation stages
in Scientific Reports
| Title | Single-cell RNAseq of bovine skin biopsy |
| Description | Raw single-cell RNAseq sequencing data from the bovine skin biopsy collected from healthy and DD infected heifers is generated using 10X genomics system and Illumina platform. Foot skin biopsies were collected from healthy (n=2) and chronic DD-infected (n=2) heifers in the sterile DPBS (with serum) on ice and immediately transferred to the lab. The biopsies were washed thrice with sterile DPBS and then incubated overnight in 5mg/ml Dispase II in HBSS at 4C. The epidermis was then peeled off and tissue was minced with scalpel. The mixture was transferred to enzyme cocktail consisting of 3.3mg/ml Collagenase IV, 3.3mg/ml Dispase II, and 1.5mg/ml DNase I in 0.25% Trypsin-EDTA and incubated for 90min at 37°C with shaking. Equal volume of complete DMEM (+10%FBS, +1% Pen/Strep) was added to inactivate enzymes and the suspension was passed through 70 µm cell strainer/filter. The tubes and filter were rinsed with complete DMEM (+10%FBS, +1% Pen/Strep) to collect all the cells. The filtrate was then passed through 40 µm cell strainer/filter to remove smaller debris. Filtrate was centrifuged at 500g for 10 minutes at 4C and cell pellet was resuspended in 10 ml DPBS (0.1% BSA). The mixture was again centrifuged at same condition and cell pellet was resuspended in 1 ml DPBS (1% BSA). The resulting mixture was processed with Dead cell removal kit (Miltenyi Biotec) and final cell pellet was resuspended in 500ul DPBS (+1%BSA). The cells were counted immediately on Invitrogen automated cell counter and appropriate number of cells were loaded in 10X genomics system (10X Chromium controller). For the processed samples, around 600 - 900 cells/ul were recovered and processed to target 10000 cells. The libraries were prepared following protocol from Chromium Next GEM Single Cell 3' Reagent Kits v3.1 guide and sequenced on Illumina NovaSeq X Plus. Between 580 to 723 million reads were generated for each samples. Prior to finalising the protocol to generate single cell suspension, 3 protocols were tested under different conditions. Protocol 1 as described above was finalised to work best. Other variants with above protocol included testing different sizes of biopsies, incubation time for initial dispase solution and incubation time with enzyme cocktail. Protocol 2 was based on sequential incubation of biopsies in different enzymes solutions of dispase, collagenase and then trypsin. Protocol 3 was entirely based on Miltenyi Biotec's procedure of using Ctubes on GentleMACS instrument with Program h_skin_01. After each protocol cells were collected/resuspended in 500ul DPBS (+1%BSA) and counted on Invitrogen Automated Cell counter. The number of cells and the viability measures were used to judge the performance of each protocol and Protocol 1 was better among all and hence used for all sample processing. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2025 |
| Provided To Others? | No |
| Impact | The data is still being analysed and would be made publicly available for use by other researchers. |
| Title | Single-nuclei RNAseq of bovine skin biopsy |
| Description | Raw single-nuclei RNAseq sequencing data from the bovine skin biopsy collected from healthy and DD infected heifers is generated using 10X genomics system and Illumina platform |
| Type Of Material | Database/Collection of data |
| Year Produced | 2025 |
| Provided To Others? | No |
| Impact | The data is still being analysed and would be made publicly available for use by other researchers. |
| Description | Collaboration with SRUC and University of Liverpool |
| Organisation | Scotland's Rural College |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This project is a Joint application between SRUC, University of Liverpool and RVC. RVC is responsible for the post-genomic functional work of this project. |
| Collaborator Contribution | This project is a Joint application between SRUC, University of Liverpool and RVC. University of Liverpool and SRUC are responsible for the collection of data, immunological profile of animals and genetic analysis. |
| Impact | Multi-disciplinary collaboration among vet, animal scientists, geneticists, immunologists, computational biologists, molecular biologists. |
| Start Year | 2018 |
| Description | Collaboration with SRUC and University of Liverpool |
| Organisation | Scotland's Rural College |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This is a collaborative joint grant-RVC contributes expertise on genomics, bioinformatics, immunology, molecular genetics |
| Collaborator Contribution | Veterinary specialisation on cattle health, microbiology, animal breeding |
| Impact | Multi-disciplinary collaboration among vet, animal scientists, geneticists, bioinformaticians, microbiologists, stem cell experts, immunologists, computational biologists, molecular biologists. |
| Start Year | 2023 |
| Description | Collaboration with SRUC and University of Liverpool |
| Organisation | University of Liverpool |
| Department | Liverpool Centre for Genomic Research |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This is a collaborative joint grant-RVC contributes expertise on genomics, bioinformatics, immunology, molecular genetics |
| Collaborator Contribution | Veterinary specialisation on cattle health, microbiology, animal breeding |
| Impact | Multi-disciplinary collaboration among vet, animal scientists, geneticists, bioinformaticians, microbiologists, stem cell experts, immunologists, computational biologists, molecular biologists. |
| Start Year | 2023 |
| Description | Collaboration with SRUC and University of Liverpool |
| Organisation | University of Liverpool |
| Department | School of Veterinary Science Liverpool |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This project is a Joint application between SRUC, University of Liverpool and RVC. RVC is responsible for the post-genomic functional work of this project. |
| Collaborator Contribution | This project is a Joint application between SRUC, University of Liverpool and RVC. University of Liverpool and SRUC are responsible for the collection of data, immunological profile of animals and genetic analysis. |
| Impact | Multi-disciplinary collaboration among vet, animal scientists, geneticists, immunologists, computational biologists, molecular biologists. |
| Start Year | 2018 |
| Description | Collaboration with SRUC and University of Liverpool |
| Organisation | University of Liverpool |
| Department | School of Veterinary Science Liverpool |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This is a collaborative joint grant-RVC contributes expertise on genomics, bioinformatics, immunology, molecular genetics |
| Collaborator Contribution | Veterinary specialisation on cattle health, microbiology, animal breeding |
| Impact | Multi-disciplinary collaboration among vet, animal scientists, geneticists, bioinformaticians, microbiologists, stem cell experts, immunologists, computational biologists, molecular biologists. |
| Start Year | 2023 |
| Description | A presentation titled "Exploring Host-Pathogen Dynamics in Bovine Digital Dermatitis: In Vivo and In Vitro Studies" during the G2P in a dish Workshop |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | The EuroFAANG G2P Workshop brought together top researchers and experts to explore the latest advancements in genotype-to-phenotype research for farmed animals. The event featured talks on genome editing, biobanking, single-cell analysis, and in vitro models, with hands-on sessions and discussions shaping the future of animal genetics research. From theoretical talks to practical applications, participants gained valuable insights into technologies and methodologies. The work was presented as a poster to demonstrate the ongoing research work within the project. |
| Year(s) Of Engagement Activity | 2025 |
| URL | https://g2p-in-a-dish.workshop.inrae.fr/ |
| Description | Participation in an open day event - Night at the Vet college |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Schools |
| Results and Impact | Participation in an open day event at the RVC presenting past and ongoing research projects from the group explaining how genetics research can improve animal health and welfare. The event was designed to engage future students and their families as well as young children to RVC research activities and inspire future scientists. |
| Year(s) Of Engagement Activity | 2024 |
| URL | https://www.rvc.ac.uk/study/rvc-is-open-for-all/visit-us/night-at-the-vet-college |