Background

Canine cutaneous and renal glomerular vasculopathy (CRGV), also known as Alabama Rot, is a rare but often fatal disease in dogs, characterised by skin lesions and acute kidney injury (AKI) due to thrombotic microangiopathy (TMA). Despite ongoing research since 2012, the precise cause has remained unknown. Our previous PhD studentship at Bristol Veterinary School investigated bacterial involvement in CRGV, ruling out previously suggested pathogens and instead identified a bacterium not normally found in the UK  as a highly prevalent bacterium in the tissue of affected dogs. Through a series of phenotypic and genotypic analyses, the study outlined important characteristics of the bacteria found in CRGV cases. This includes: organ distribution, growth at various temperatures including body temperature, haemolytic activity, cytotoxicity against canine kidney cells, possible biofilm formation, antimicrobial susceptibility profiles, and genetic analysis within a broader collection of related strains. The findings showed that the bacteria from CRGV cases are multidrug resistant, exhibit different levels of virulence and virulence traits, contain a large clonal group that appears novel to the UK and is different to previous clinical isolates.

While this work provides the discovery and initial characterisation of a potentially novel pathogen, extensive research is still needed. Microbiological and genomic techniques should be used to identify virulence mechanisms and uncover unique biomarkers for diagnostic tests. In addition, by understanding traits further, such as whether it produces a particular toxin, appropriate treatments could be developed, potentially including novel therapies, given its multidrug-resistant nature.

Aim/objectives

The work has two aims:

Firstly to analyse in detail the full genome sequence of all 25 isolates of the causative bacterium to identify a unique gene target that will identify this bacterium and can be used as a PCR diagnostic test for identification of the bacterium in CRGV cases. This test will be offered on the open market.

Second, to analyse the sequences to identify toxins carried by the bacterium, to purify these toxins or express them in E. coli and to identify the cells types that they bind to in order to determine whether these represent canine kidney and/or skin toxins. This work will be carried out as a collaboration with UoB’s academic Renal Unit. Identification of the toxins would enable production of an antitoxin or vaccine treatment for CRGV.

Methods

Bacteria have already been sequenced and this used to determine the presence of a novel clonal group. This sequence data will be aligned against other genes to identify novel sequences in the target strains and these used to produce sets of PCR primers which will be screened and validated against other bacteria. Dr Cogan’s diagnostic laboratory routinely designs these type of tests for other bacteria, and the test can be offered to vets for diagnostic use through Langford Vets’ diagnostic service as a not for profit service.
The same sequence data will be used to identify putative toxin genes using the Toxinome database and a custom analysis pipeline. Culture supernatant will be purified on an AKTA using size-exclusion chromatography and the fractions assayed for binding to canine kidney cell lines and primary skin and kidney cells. Fractions induing cell death will be analysed by mass spectroscopy to identify proteins present and these mapped back to toxin genes. This equipment is available at the vet school and central University of Bristol. Should this technique not identify pure proteins toxin genes will be cloned into an E coli expression vector, purified on an AKTA using affinity chromatography and then assayed as above. 

Funding requirement

Total of £49,932.13 comprising:

£44,032.13 - Dr Helen Howshall salary as PDRA plus technical support
£5000 - consumables for AKTA work and cell studies
£900 - travel and sample transport between Langford, central Bristol and Academic Renal Unit in North Bristol, and conference presentation.

Recipient of funds
 
Bristol Veterinary School – Dr Tristan Cogan. No salary or overheads - funds as above will go directly to this project.

Timeline
 
First quarter 2026 for 12 months.

Where and when results published
 
At end of project – toxin work published in a journal such as Veterinary Microbiology. Results will also be presented to a veterinary clinical audience at a conference such as ECVM.