Professor Nicholas Evans B.Sc., Ph.D., F.H.E.A.

Leptospirosis is a global, severe infectious disease affecting several different species including ruminants and man. Cattle infection with leptospires results in severe economic losses, food security impact and substantial antimicrobial use, as well as increased zoonotic spread. Bovine leptospirosis (BL) vaccines are available although there are constraints inhibiting use in tropical regions where most disease burden is prevalent including limited range of specificity and cold chain transport and storage issues.
We have characterised several leptospiral surface proteins including adhesion of host molecules to the bacterial cell surface proteins and interaction with the host immune system. Subsequently we have also identified a diversity in function across surface protein orthologs across genomospecies and serovars. Our most recent research is towards engineering more thermostable and efficacious leptospire vaccines. Vaccines are considered key mechanisms to reduce AMR. Making vaccines more broadly protective, easily accessible and financially affordable can only increase uptake globally, therefore decreasing antibiotic use and AMR.

Funding:
BBSRC RM Grant: Dissecting cell surface protein diversity to enhance leptospiral vaccine efficacy. N. Evans (PI), D. Rigden, S. Carter & H. Crosby-Durrani.
BBSRC/MRC/GCRF Bactivac Pump Priming Award: Enhancing the efficacy of bovine leptospiral vaccines. N. Evans (PI), J. Derrick (University of Manchester), S. Carter, C. Fenner (Afrigen, South Africa) & Intan Kamaruzaman (Universiti Malaysia Kelantan, Malaysia).
Malaysian government funded PhD Studentship: Delineating host-pathogen interaction of pathogenic Leptospira spp. Supervisors: N. Evans (Lead) & S. Carter, Student: Intan Kamaruzaman.

The majority of evidence clearly identifies bacteria known as 'Treponema' as the common cause of BDD. In previous studies we clarified the association of these bacteria with BDD and characterised these causal bacteria into three distinct groups. We developed novel PCR diagnostics for the DD treponemes which have widely been applied for diagnostic testing of the disease (1). These PCR diagnostics allowed us to track the continued emergence of the disease in new hosts including sheep, beef cattle, and wild elk and in new countries (Ireland, Germany, USA) (2-4). Multi locus sequence typing of a large panel of strains (n=121) from multiple hosts and continents demonstrated that the same bacterial strains appear to be transmitted between different host species although human strains appear to have evolved separately (5). We have also implicated the GI tract as an infection reservoir in dairy and beef cattle and sheep (6).
Characterisation of the bacteria considered causal of these diseases and clarification of transmission routes should enable future control methods to tackle this severe infectious disease of ruminants.

Outputs:
1. Evans, N. J., Brown, J. M., Demirkan, I., Singh, P., Getty, B., Timofte, D., . . . Carter, S. D. (2009). Association of Unique, Isolated Treponemes with Bovine Digital Dermatitis Lesions. Journal of Clinical Microbiology, 47(3), 689-696. DOI: 10.1128/JCM.01914-08 (DOI: 10.1128/JCM.01914-08)
2. Sullivan, L. E., Clegg, S., Angell, J. W., Newbrook, K., Blowey, R. W., Carter, S., . . . Evans, N. (2015). High-Level Association of Bovine Digital Dermatitis Treponema spp. with Contagious Ovine Digital Dermatitis Lesions and Presence of Fusobacterium necrophorum and Dichelobacter nodosus. Journal of Clinical Microbiology, 53(5), 1628-1638. DOI: 10.1128/JCM.00180-15 (DOI: 10.1128/JCM.00180-15)
3. Clegg, S., Mansfield, K., Newbrook, K., Sullivan, L., Blowey, R., Carter, S., & Evans, N. 2015 Isolation of digital dermatitis treponemes from hoof lesions in wild elk in Washington State, USA. Journal of Clinical Microbiology, 53(1):88-94. DOI: 10.1128/JCM.02276-14 (DOI: 10.1128/JCM.02276-14)
4. Tegtmeyer, P. C., Staton, G. J., Evans, N. J., Rohde, J., Punsmann, T. M., & Ganter, M. (2020). First cases of contagious ovine digital dermatitis in Germany. Acta Veterinaria Scandinavica, 62(1). DOI: 10.1186/s13028-020-00544-0 (DOI: 10.1186/s13028-020-00544-0)
5. Clegg, S. R., Carter, S. D., Birtles, R. J., Brown, J. M., Hart, C. A., & Evans, N. J. (2016). Multilocus Sequence Typing of Pathogenic Treponemes Isolated from Cloven-Hoofed Animals and Comparison to Treponemes Isolated from Humans. Applied and Environmental Microbiology, 82(15), 4523-4536. DOI: 10.1128/AEM.00025-16 (DOI: 10.1128/AEM.00025-16)
6. Sullivan, L. E., Carter, S. D., Duncan, J. S., Grove-White, D. H., Angell, J. W., & Evans, N. J. (2015). The Gastrointestinal Tract as a Potential Infection Reservoir of Digital Dermatitis-Associated Treponemes in Beef Cattle and Sheep. Applied and Environmental Microbiology, 81(21), 7460-7469. DOI: 10.1128/AEM.01956-15 (DOI: 10.1128/AEM.01956-15)

Funding:
BBSRC RM Grant: Dissecting the molecular diversity and commonality of the bovine digital dermatitis treponemes. New Investigator Award: N. Evans (PI).
BBSRC RM Grant: Host-Pathogen interactions of treponemes and hoof tissues in digital dermatitis: How does infection lead to lameness. S. Carter (PI), R. Birtles, N. Evans (Researcher Co-I)
AHDB Dairy/DairyCo Funded Studentship: Identifying how digital dermatitis is transmitted between dairy cows. Supervisors: N. Evans (Lead), R. Murray & S. Carter, Student: Jennifer Bell.
AHDB Beef and Lamb/HCC/QMS Funded Studentship: Identifying digital dermatitis infection reservoirs in beef cattle and sheep. Supervisors: N. Evans (Lead), J. Duncan, D. Grove-White & S. Carter, Student: Leigh Sullivan.


Disinfection of foot trimming hoof knives to reduce transmission of digital dermatitis.

During initial surveys of BDD treponeme infection reservoirs we struggled to find substantial evidence of infection reservoirs other than infected feet (1) whilst implicating some contribution of the ruminant gastrointestinal tract (1,2). Subsequently, akin to the human treponematoses yaws, which is considered transmitted by touch, we were able to demonstrate that foot trimming equipment which can quickly be moved from cattle hoof to hoof without disinfection are frequently contaminated with digital dermatitis treponemes (3).
Subsequently, using the treponemal culture and diagnostic methods we had previously developed (4), we showed that DD treponemes could be transmitted on hoof trimming knives and gloves (5, 6). We subsequently used both in vitro and field-based studies to assess disinfectant efficacy (6, 7). This has led to the creation of an industry published evidenced based disinfection protocol to prevent the transmission of disease between and within farms (8). This protocol has now been adopted by both national (UK) and international dairy professional/vets and farmers (9) as well as having led to a better awareness of hygiene practices during foot trimming (9).

Outputs:
1. Evans, N. J., Timofte, D., Isherwood, D. R., Brown, J. M., Williams, J. M., Sherlock, K., Carter, S. D. (2012). Host and environmental reservoirs of infection for bovine digital dermatitis treponemes. Veterinary Microbiology, 156(1-2), 102-109. DOI: 10.1016/j.vetmic.2011.09.029 (DOI: 10.1016/j.vetmic.2011.09.029)
2. Sullivan, L. E., Carter, S. D., Duncan, J. S., Grove-White, D. H., Angell, J. W., & Evans, N. J. (2015). The Gastrointestinal Tract as a Potential Infection Reservoir of Digital Dermatitis-Associated Treponemes in Beef Cattle and Sheep. Applied and Environmental Microbiology, 81(21), 7460-7469. DOI: 10.1128/AEM.01956-15 (DOI: 10.1128/AEM.01956-15)
3. Sullivan, L. E., Blowey, R. W., Carter, S. D., Duncan, J. S., Grove-White, D. H., Page, P., . . . Evans, N. J. (2014). Presence of digital dermatitis treponemes on cattle and sheep hoof trimming equipment. VETERINARY RECORD, 175(8), 201-+. DOI: 10.1136/vr.102269 (DOI: 10.1136/vr.102269)
4. Evans, N. J., Brown, J. M., Demirkan, I., Singh, P., Getty, B., Timofte, D., . . . Carter, S. D. (2009). Association of Unique, Isolated Treponemes with Bovine Digital Dermatitis Lesions. Journal of Clinical Microbiology, 47(3), 689-696. DOI: 10.1128/JCM.01914-08 (DOI: 10.1128/JCM.01914-08)
5. Angell, J. W., Clegg, S. R., Grove-White, D. H., Blowey, R. W., Carter, S. D., Duncan, J. S., & Evans, N. J. (2017). Survival of contagious ovine digital dermatitis (CODD)-associated treponemes on disposable gloves after handling CODD-affected feet. VETERINARY RECORD, 181(4), 89. DOI: 10.1136/vr.104228 (DOI: 10.1136/vr.104228)
6. Gillespie, A., Carter, S. D., Blowey, R. W., & Evans, N. (2020). Survival of bovine digital dermatitis treponemes on hoof knife blades and the effects of various disinfectants. The Veterinary Record, 186(2). DOI: 10.1136/vr.105406 (DOI: 10.1136/vr.105406)
7. Gillespie, A. V., Carter, S. D., Blowey, R. W., Staton, G. J., & Evans, N. J. (2020). Removal of bovine digital dermatitis-associated treponemes from hoof knives after foot-trimming: a disinfection field study. BMC VETERINARY RESEARCH, 16(1). DOI: 10.1186/s12917-020-02552-8 (DOI: 10.1186/s12917-020-02552-8)
8. https://ahdb.org.uk/knowledge-library/cattle-foot-trimming-equipment-disinfection
9. Gillespie, A. V., Carter, S. D., Blowey, R. W., Staton, G. J., Walsh, T. R., & Evans, N. J. (2021). Measuring the impact of bovine digital dermatitis research on knowledge and practice of biosecurity during cattle foot-trimming. JOURNAL OF DAIRY RESEARCH, 88(1), 60-63. DOI: 10.1017/S0022029921000170 (DOI: 10.1017/S0022029921000170)

Funding:
BBSRC DTP CASE Studentship with AHDB Dairy: Preventing transmission of bovine digital dermatitis. Supervisors: N. Evans (Lead) & S. Carter, Student: Amy Gillespie.
BBSRC RM Grant: Dissecting the molecular diversity and commonality of the bovine digital dermatitis treponemes. New Investigator Award: N. Evans (PI).
BBSRC RM Grant IPA with AHDB Beef and Lamb and HCC: Unravelling the aetiology of contagious ovine digital dermatitis. N. Evans (PI), J. Duncan, D. Grove-White & S. Carter.
AHDB Dairy/DairyCo Funded Studentship: Identifying how digital dermatitis is transmitted between dairy cows. Supervisors: N. Evans (Lead), R. Murray & S. Carter, Student: Jennifer Bell.
AHDB Beef and Lamb/HCC/QMS Funded Studentship: Identifying digital dermatitis infection reservoirs in beef cattle and sheep. Supervisors: N. Evans (Lead), J. Duncan, D. Grove-White & S. Carter, Student: Leigh Sullivan.

In a recent study, we collected entire genome and proteome data for several treponemal bacteria considered causal of bovine digital dermatitis (BDD). We also generated genomes for bovine gastrointestinal treponemes and relevant human treponemes and investigated the distribution of relevant virulence factors across the range of treponemes investigated.
Using the genomic data we completed a reverse vaccinology approach identifying a diagnostic antigen and potential vaccine candidates (1).
Further comparative genomic analyses identified production of a novel mannuronic acid sugar as central to BDD treponeme pathogenesis (2). Moreover, relevant survival characteristics were determined which appear to be key to these pathogens surviving on the surface of skin and within the farm environment. Recombinant outer membrane protein generation from key orthologs across the different BDD treponemes determined that most enable host attachment, with considerable functional diversity and redundancy determined (2).
We developed cattle skin fibroblast and keratinocyte models and undertook transcriptomics of cattle skin fibroblasts which had been challenged with the BDD treponemes identifying key inflammatory markers involved in disease pathogenesis (3). Moreover, comparing challenge with pathogenic and commensal treponemes we demonstrated that BDD treponemes appear to be able to immune-evade the host response (3).

These studies have enhanced understanding of the respective pathogens and should allow for future vaccine, diagnostic and treatment studies to better protect from this severe disease of ruminants.

Key Outputs:
1. Staton, G. J., Carter, S. D., Ainsworth, S., Mullin, J., Smith, R. F., & Evans, N. J. (2020). Putative ß-Barrel Outer Membrane Proteins of the Bovine Digital Dermatitis-Associated Treponemes: Identification, Functional Characterization, and Immunogenicity. Infection and immunity, 88(5).DOI: 10.1128/IAI.00050-20 (DOI: 10.1128/IAI.00050-20)
2. Newbrook, K., Carter, S. D., Crosby-Durrani, H., & Evans, N. J. (2021). Challenge of Bovine Foot Skin Fibroblasts With Digital Dermatitis Treponemes Identifies Distinct Pathogenic Mechanisms. Frontiers in Cellular and Infection Microbiology, 10. DOI: 10.3389/fcimb.2020.538591 (DOI: 10.3389/fcimb.2020.538591)
3. Staton, G. J., Clegg, S. R., Ainsworth, S., Armstrong, S., Carter, S. D., Radford, A. D., Darby, A., Hall, N., Wastling, J. & Evans, N. J. (2021). Dissecting the molecular diversity and commonality of bovine and human treponemes identifies key survival and adhesion mechanisms. PLOS PATHOGENS, 17(3). DOI: 10.1371/journal.ppat.1009464 (DOI: 10.1371/journal.ppat.1009464)

Funding:
BBSRC RM Grant: Dissecting the molecular diversity and commonality of the bovine digital dermatitis treponemes. New Investigator Award: N. Evans (PI).
BBSRC Studentship: Dissecting host-pathogen interactions in bovine digital dermatitis. Supervisors: N. Evans (Lead) & S. Carter, Student: K. Newbrook.



Unravelling the aetiology of contagious ovine digital dermatitis.

We tracked the clinical progression as well as metagenomic and qPCR analyses of infected sheep throughout a natural outbreak of contagious ovine digital dermatitis, an important infectious disease causing lameness in sheep. These analyses further dissect the cause of this important disease and enable identification of key pathogens and infection reservoirs (1,2). We further clarified the contribution of footrot within the aetiology of contagious ovine digital dermatitis (1) which has underpinned advice to the sheep industry lameness manual. We demonstrated that gloves become contaminated with the associated pathogens during foot trimming and describe best practice for disinfection (3). We have also been involved in reporting the first outbreak of CODD in mainland Europe (4) and published a manuscript dissecting the aetiology of a novel ulcerative leg lesion of sheep (5).

Funding
BBSRC RM Grant IPA with AHDB Beef and Lamb and HCC: Unravelling the aetiology of contagious ovine digital dermatitis. PIs: N. Evans & J. Duncan, D. Grove-White & S. Carter.

Outputs
1. Duncan, J. S., Angell, J. W., Richards, P., Lenzi, L., Staton, G. J., Grove-White, D., . . . Evans, N. J. (2021). The dysbiosis of ovine foot microbiome during the development and treatment of contagious ovine digital dermatitis. Animal microbiome, 3(1), 19. doi:10.1186/s42523-021-00078-4 DOI: 10.1186/s42523-021-00078-4
2. Staton, G. J., Angell, J. W., Grove-White, D., Clegg, S. R., Carter, S. D., Evans, N. J., & Duncan, J. S. (2021). Contagious Ovine Digital Dermatitis: A Novel Bacterial Etiology and Lesion Pathogenesis. FRONTIERS IN VETERINARY SCIENCE, 8. doi:10.3389/fvets.2021.722461 DOI: 10.3389/fvets.2021.722461
3. Angell, J. W., Clegg, S. R., Grove-White, D. H., Blowey, R. W., Carter, S. D., Duncan, J. S., & Evans, N. J. (2017). Survival of contagious ovine digital dermatitis (CODD)-associated treponemes on disposable gloves after handling CODD-affected feet. VETERINARY RECORD, 181(4), 89. DOI: 10.1136/vr.104228
4. Tegtmeyer, P. C., Staton, G. J., Evans, N. J., Rohde, J., Punsmann, T. M., & Ganter, M. (2020). First cases of contagious ovine digital dermatitis in Germany. Acta Veterinaria Scandinavica, 62(1). DOI: 10.1186/s13028-020-00544-0
5. Staton, G. J., Crosby-Durrani, H., Roberts, G., Duncan, J. S., Seechurn, N., Blowey, R., & Evans, N. J. (2020). Novel ulcerative leg lesions in yearling lambs: Clinical features, microbiology and histopathology. Veterinary Microbiology, 247. doi:10.1016/j.vetmic.2020.108790 DOI: 10.1016/j.vetmic.2020.108790