Dissecting the molecular diversity of bovine digital dermatitis treponemes.

Bovine Digital Dermatitis (BDD) is a lameness of cattle present in the majority of dairy herds in the UK and reported throughout the world. This lameness is the result of inflamed lesions between the heel bulbs of the rear feet. The disease is of particular significance because it is very painful for the animal affected resulting in poor animal welfare. Despite some response to commonly used antibiotics, lesions frequently reappear. The disease is an important global food security issue because it causes reduction in milk production and poor reproductive performance in dairy cows. Furthermore, with the recent cost to the dairy industry in the UK likely to be as much as 26 million pounds per year, it has a negative effect the UK's economic competitiveness. The disease has now been identified in sheep, where it is known as Contagious Ovine Digital Dermatitis (CODD) with lesions more severe than seen in cattle with frequent reports of complete hoof loss.
BDD has been identified as an infectious disease with a bacterial cause. The majority of evidence clearly identifies bacteria known as 'Treponema' as the common cause of BDD. Previous studies at the University of Liverpool have clarified the association of these bacteria with BDD and characterised these causal bacteria into three distinct groups. These studies have allowed for the isolation of 70 treponemes from BDD lesions, a greater number than any other research groups working on this disease worldwide. This strain collection is an invaluable resource for investigating how these organisms produce such severe disease in cattle and sheep and how the infection may so readily be transmitted between animals.
The proposed research in this current grant application will use this large panel of isolates to investigate how these organisms cause the disease in cattle and whether novel vaccines or treatments can be developed. The three distinct groups of bacteria (treponemes) are very different from one another in terms of their genetics and their actual characteristics yet they are all able to colonise the bovine hoof and cause disease. Whilst the different treponeme groups may be quite diverse, to be able to share the same 'niche', they may still share very similar machinery to be able to grow, survive and damage the cow. This common machinery is likely to be proteins exhibited on the surface of the cell which are responsible for binding or damaging the host and if shared across all BDD treponemes may have potential as vaccine targets. Identification of these key bacterial proteins and determining whether they interact with the host immune system allows for identification of potential vaccine components and thus enable a strategy to prevent disease in cattle and sheep herds.
At the University of Liverpool, there have been major investments in the current cutting edge technologies of genomics and proteomics and with these facilities it is now possible to perform substantial experiments not previously possible. For example, whole genomes of these treponemes can now be generated in a relatively short time. This study proposes to use this cutting edge sequencing technology to investigate, on a large scale all the predicted disease causing characteristics of the bacteria. To confirm that the genes identified are in fact expressed the proteomics approaches will be used to identify all the proteins both inside the treponemes and those on the surface. Furthermore cattle sera will be incubated with the bacterial cell surface proteins to identify which cell surface proteins are bound to by antibodies and therefore to actually identify which are detected by the cattle's immune system. These studies will help characterise the causal bacteria and further aid in potential vaccine design. By investigating BDD using the various methods described above, we should improve understanding of the disease and contribute towards the eradication of this painful and expensive disease.

Bovine digital dermatitis (BDD) is a lameness in cattle caused by foot lesions that are exceptionally painful and result in an important animal welfare issue. The disease has significant economic implications resulting from reduction in milk yield and reproductive performance. The disease is present worldwide, endemic in UK dairy herds and costs the UK 26 million pounds per year.
This proposal aims to further understand the disease by investigating the shared and unique cell surface proteins of the BDD treponemes and to identify whether they are antigenic. The main resource for this study is the current panel of treponeme isolates (70+) as well as previously collected draft genome sequences.
Genome sequences will be collected for 2x bovine commensal gastrointestinal (GI) tract treponemes using a GS-FLX platform sequencer. New genome sequences and previously collected genome sequences (3x BDD and 2x human nearest relative treponemes) will be completed and compared and analysed using ACT to identify genes encoding shared or unique cell surface protein.
The seven treponemes will be grown and various extractions collected using French pressure cell (membrane associated and soluble) or Triton X-114 (outer membrane enriched) isolations. Extractions will be analysed by MUDPIT analysis as well as 2D gel electrophoresis as recently carried out for Treponema denticola. Relevant spots on 2D gels will be picked, trypsin digested and subjected to mass spectrometer identification. To identify BDD treponeme antigens that are shared or unique the 2D gels will be probed with antisera (symptomatic or asymptomatic from BDD farm or cattle from a farm without BDD). The presence of important antigens across the large strain panel will be investigated by PCR and the respective genes sequenced. Gene diversity will be identified on structural models produced for each of the important antigens.

Bovine digital dermatitis (BDD) is an infectious disease causing lameness in dairy cattle worldwide and is of particular significance as it is extremely painful for animals involved and reduces milk yield and reproductive performance. It has been reported that 70-90% of UK dairy herds are affected by BDD with 30-60% morbidity suggesting a large number of animals suffer in the UK alone. The major benefactors of this research would be cattle as results produced could aid in eradicating BDD which in turn would stop the suffering for millions of animals worldwide. Given this disease appears to be emerging into new hosts and diseases such as sheep and pigs and new non-healing bovine horn lesions it would lead to better animal welfare for the animals involved in these diseases also.
The general public worldwide and especially in UK would benefit from this research if it is successful in identifying new vaccine candidates or effective treatments as there would be reduced cost of dairy products and therefore a better standard of living. This reduced cost would be because BDD is very expensive with costs accruing from increased hoof inspection, veterinary fees, expensive (ineffective) treatments and reduction in milk yield and reproductive performance. Farmers would benefit as this is a costly disease in terms of time and finances. Prevention or eradication of this disease would have a positive effect on farm economics both worldwide and in the UK.
There would also be environmental impact if BDD is eradicated by development of a vaccine resulting from the research. Vaccination would reduce the need for footbaths and over use of antibiotics. Footbaths typically consist of formalin or copper sulphate which are damaging to the environment. Use of antibiotic treatments can result in antibiotic residues in milk that can lead to milk withdrawal. Furthermore, overuse of antibiotics may induce antibiotic resistance in the causative and environmental bacteria, which could be damaging to the environment.
There will also be beneficiaries within the commercial sector who will benefit from this research as this project will place in the public domain information regarding novel treatments or identify antigens likely to make good vaccine components. Whilst I have no formal agreements in place with commercial concerns, there has been considerable interest from companies who have been in contact. Once I begin to identify relevant vaccine candidates and potential treatments I will approach companies with whom I have had previous contact to seek future collaborations. If I believe the treatments/strategies I identify for BDD are more widely applicable to other spirochete diseases then I will contact industry directly regards possible opportunities or will form relationships with relevant academic groups studying these diseases so as they can be further investigated.
In terms of realistic timescales for benefits to be realised from this grant, it could be considered completion of this work will have contributed to substantially underpinning future vaccine design and treatments. Therefore at the end of the grant (3 years) an industrial concern should then be able to use the information produced to develop an effective vaccine or treatment within another 2-3 years.
Staff on the project will develop a comprehensive knowledge of genomics, proteomics, veterinary infectious diseases and preliminary investigations into identifying vaccine candidates. These would benefit both the research associate and principal investigator by giving them skills both useful to academia and industry. By having research dedicated staff studying BDD at UoL we are able to support UK biotech companies better, for example I am currently helping Ridgeway Biologicals by transferring culturing skills. The continuation of such activity is helped greatly by having a larger staff base and can only be good for the UK economy and increase its global economic performance.