The role of the gut microbiota in the pathology of cutaneous and renal glomerular vasculopathy (CRGV) – a shotgun metagenomic sequencing approach
Interim Report September 2024 - produced by Jack Whitehouse

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Interim Report February 2024 - produced by Jack Whitehouse

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Hypothesis and Purpose of Project
Does the gut microbiota play a role in the disease process of CRGV? Establishing differences in the bacterial species in the gut microbiota of dogs suffering from CRGV compared to dogs suffering from Acute Kidney Injury, and Medically healthy dogs would improve our chances of identifying bacterial species that may be responsible for CRGV, either as a direct cause or as a contributing factor.
Background
The faecal microbiota encompasses a diverse range of organisms including bacteria that perform a variety of functions influencing the overall health of a host, such as nutrient metabolism, immune regulation, and host defence1. Changes in the abundance of bacterial species have previously been associated with several diseases1. For example, inflammatory bowel disease in dogs was shown to be associated with reduced bacterial diversity. We hypothesise that changes to the gut microbiota may also play a role in the disease processes of CRGV. Indeed, some bacteria such as Aeromonas hydrophilia2 and Escherichia coli3, may be involved in CRGV on the basis of either the geographical distribution of CRGV cases across wet environments4 (e.g., wet floor meadows) where A. hydrophilia is encountered, or the similarity of CRGV with Haemolytic Uraemic Syndrome (HUS) caused by certain types of E. coli in humans5. A pilot investigation by the research team at the School of Veterinary Medicine, University of Surrey, comparing the faecal microbiota of CRGV and healthy dogs, has indicated changes that require further investigation.
Objectives
- Prepare a whole-genome library for sequencing of faecal microbiota (96 samples: CRGV-affected n = 32; AKI n= 32; medically healthy n = 32). Send DNA samples to a third-party sequencing provider for shotgun metagenomic sequencing.
- Analyse data for correlative associations between bacterial species (CRGV-affected, AKI and medically healthy dogs) and compare the metadata for the study animals (e.g., age, breed, neuter status, gender, treatment, and disease outcome) to determine potential differences.
Methods
CRGV canine faecal samples were collected on a case-by-case basis by veterinary surgeons at Anderson Moores Veterinary Specialists between September 2016 and September 2020. Faecal samples from medically healthy dogs were collected between August and September 2020 through a public-recruitment campaign. Further collection of faecal samples from dogs diagnosed with oligo-anuric AKI will be carried out in partnership with Anderson Moores Veterinary Specialists and the University of Surrey, School of Veterinary Medicine Partnership Network between November 2022 and January 2023. Upon receipt of the faecal samples at the School of Veterinary Medicine, University of Surrey, the CRGV and medically healthy samples underwent DNA extraction using a commercially available DNA extraction kit, while the AKI samples will undergo DNA extraction on arrival before use in constructing the genome libraries required for the shotgun metagenomic sequencing6. The study will use DNA library preparation kits to create these libraries before sending the samples off to a third party for sequencing. Statistical analysis will be carried out to test for differences in the observed bacterial species between sample groups.
Impact
If our preliminary results on the structure of the faecal microbiota of CRGV-affected dogs are supported by the shotgun metagenomic sequencing, it may provide stronger evidence for the role of specific bacteria in CRGV. Furthermore, studying all bacterial species in the faecal microbiota of CRGV-affected dogs may identify additional unique species that could be responsible for causing CRGV and/or contributing to the severity of the disease. The potential outcomes for the proposed research may include: (1) elucidating unique pathogenic species associated with CRGV; (2) mapping the total bacterial species present in the faecal microbiota at the time samples were collected; (3) establishing how these changes may impact the host during disease through an extensive literature review; and (4) determining how the main faecal microbiota species could be targeted therapeutically to alter disease outcome.
Project Lead
The funds will be received by Prof. Roberto La Ragione, the main applicant, Head of the School of Biosciences, and Professor of Veterinary Microbiology and Pathology in the School of Veterinary Medicine at the University of Surrey. Prof. Roberto La Ragione supervises the PhD Student (Mr Jack Whitehouse; https://www.surrey.ac.uk/people/jack-whitehouse) who performed the practical aspects of the preliminary study. Prof. La Ragione is an experienced researcher, with expertise that includes veterinary microbiology, with a particular interest in host-microbe interactions and the role of natural microflora in health and disease. Recent publications can be found at https://www.surrey.ac.uk/people/roberto-la-ragione. This study will further complement the ARRF funded-PhD studentship at the University of Surrey, entitled “The role of the microbiome and circulating endothelial cells in the pathobiology of Cutaneous and Renal Glomerular Vasculopathy (CRGV)” since the core purpose of the PhD is to investigate the role that the gut microbiota plays in the disease process of CRGV. This PhD project is supervised by Prof. Roberto La Ragione; Prof. Mark Chambers, Head of the Department of Microbial Sciences, School of Biosciences and Professor of Microbiology and Disease Intervention; Dr Joy Leng, Research Fellow (School of Veterinary Medicine), and Mr David Walker, Director at Anderson Moores Veterinary Specialists.
Where and when will the results be published
The timeline for the project is estimated to be seven months. The results will be disseminated through the ARRF and submitted for publication in a high-impact veterinary journal, such as BMC Animal Microbiome. The findings will also be presented at pertinent seminars and conferences, such as BSAVA Congress and the Microbiology Society annual conference.
Funding requirements
Consumables/Staff time Cost
Whole genome sequencing £6,000
Library preparation kits £5,894
General consumables and staff time £1,106
Total funding requested £13,000
Whole genome sequencing £6,000
Library preparation kits £5,894
General consumables and staff time £1,106
Total funding requested £13,000
References
- Pilla, R., et al. (2019) ‘The Role of the Canine Gut Microbiome and Metabolome in Health and Gastrointestinal Disease’, Front Vet Sci, 14 (6), pp. 498. doi:10.3389/fvets.2019.00498.
- Macdonald, F. A. (2015) ‘Role of Aeromonas hydrophila in CRGV in Dogs’, Vet Rec, 177 (23), pp. 602. doi:10.1136/vr.h6667.
- Walker, J. J. A., et al. (2021) ‘Clinicopathological features of cutaneous and renal glomerular vasculopathy in 178 dogs’, Vet Rec, 189 (4). doi: 10.1002/vetr.72.
- Stevens, K. B., et al. (2018) ‘Spatiotemporal patterns and agroecological risk factors for cutaneous and renal glomerular vasculopathy (Alabama Rot) in dogs in the UK’, Vet Rec, 183 (16), pp. 502. doi: 10.1136/vr.104892.
- Mayer, C. L., et al. (2012) ‘Shiga Toxins and the pathophysiology of hemolytic uremic syndrome in humans and animals’, Toxins, 8 (11). doi: 10.3390/toxins4111261.
- Quince, C., et al. (2017) ‘Shotgun metagenomics, from sampling to analysis’, Nat Biotechnol, 35 (9), pp. 833 – 44. doi: 10.1038/nbt.3935.