The dysbiosis of ovine foot microbiome during the development and treatment of contagious ovine digital dermatitis.

BACKGROUND: Contagious Ovine Digital Dermatitis (CODD) is an emerging and common infectious foot disease of sheep which causes severe welfare and economic problems for the sheep industry. The aetiology of the disease is not fully understood and control of the disease is problematic. The aim of this study was to investigate the polybacterial aetiopathogenesis of CODD and the effects of antibiotic treatment, in a longitudinal study of an experimentally induced disease outbreak using a 16S rRNA gene amplicon sequencing approach. RESULTS: CODD was induced in 15/30 experimental sheep. During the development of CODD three distinct phenotypic lesion stages were observed. These were an initial interdigital dermatitis (ID) lesion, followed by a footrot (FR) lesion, then finally a CODD lesion. Distinct microbiota were observed for each lesion in terms of microbial diversity, clustering and composition. Porphyromonadaceae, Family XI, Veillonellaceae and Fusobacteriaceae were significantly associated with the diseased feet. Veillonellaceae and Fusobacteriaceae were most associated with the earlier stages of ID and footrot rather than CODD. Following antibiotic treatment of the sheep, the foot microbiota showed a strong tendency to return to the composition of the healthy state. The microbiota composition of CODD lesions collected by swab and biopsy methods were different. In particular, the Spirochaetaceae family were more abundant in samples collected by the biopsy method, suggesting that these bacteria are present in deeper tissues of the diseased foot. CONCLUSION: In this study, CODD presented as part of a spectrum of poly-bacterial foot disease strongly associated with bacterial families Porphyromonadaceae, Family XI (a family in Clostridiales also known as Clostridium cluster XI), Veillonellaceae and Fusobacteriaceae which are predominately Gram-negative anaerobes. Following antibiotic treatment, the microbiome showed a strong tendency to return to the composition of the healthy state. The composition of the healthy foot microbiome does not influence susceptibility to CODD. Based on the data presented here and that CODD appears to be the severest end stage of sheep infectious foot disease lesions, better control of the initial ID and FR lesions would enable better control of CODD and enable better animal welfare.

Removal of bovine digital dermatitis-associated treponemes from hoof knives after foot-trimming: a disinfection field study.

BACKGROUND: Bovine digital dermatitis (BDD) is an infectious foot disease found commonly in dairy herds. Foot-trimming is an important husbandry procedure for reducing the ensuing lameness; however, epidemiological, and microbiological studies have identified this as a risk activity for transmitting BDD. Three disinfectants have previously been identified in laboratory work as effective for removing viable BDD-associated Treponema spp., from hoof knife blades. The present study enrolled 133 dairy cattle with BDD lesions, and swabbed hoof knife blades before and after foot-trimming, and after knife disinfection with one of three disinfectants (1:100 FAM30®, 2% Virkon® and 2% sodium hypochlorite) to assess their efficacy under field conditions. RESULTS: Detection of BDD treponeme phylogroup DNA was undertaken by direct PCR of swabs, and viable treponemes were detected by PCR of swab cultures after 6 weeks' incubation. Where hoof knives did not contact the lesion, BDD-associated treponemes were detected after foot-trimming in 12/22 (54.5%) cases by direct PCR and 1/22 (4.5%) cases by PCR of cultured organisms. Where contact was made with the lesion, 111/111 (100%) samples taken after trimming were positive by direct PCR and 47/118 (39.8%) were positive by culture PCR. Viable organisms were identified in cultures from lesion stages M2, M3, M4 and M4.1. No viable organisms were detected after disinfection of hoof knives. CONCLUSIONS: Hoof knives post-trimming were frequently contaminated with BDD-associated treponeme DNA. Viable organisms were identified in cultures whether contact had been made between hoof knife and lesion or not, although contact clearly increased the frequency of detection of viable organisms. The three disinfectants tested were effective for removing viable organisms. The disinfection protocol used in this study should therefore be considered reliable for adoption as standard industry practice.

Novel ulcerative leg lesions in yearling lambs: Clinical features, microbiology and histopathology.

Here we report an outbreak of an atypical, ulcerative dermatitis in North Country mule lambs, located in South Gloucestershire, UK. The lesions, which appeared to be contagious, occured between the coronary band and the carpal joint as a focal, well demarcated, circular, ulcerative dermatitis. Histopathological examination of the lesion biopsies revealed areas of ulceration, epidermal hyperplasia, suppurative dermatitis and granulation tissue. Clumped keratohyalin granules and intracellular keratinocyte oedema (ballooning degeneration) were evident within lesion biopsies, consistent with an underlying viral aetiology. A PCR-based microbiological investigation failed to detect bovine digital dermatitis-associated treponeme phylogroups, Dichelobacter nodosus, Staphylococcus aureus, Dermatophilus congolensis or Chordopoxvirinae virus DNA. However, 3 of the 10 (30 %) and 6 of 10 (60 %) lesion samples were positive for Fusobacterium necrophorum and Streptococcus dysgalactiae DNA, respectively. Contralateral limb swabs were negative by all standard PCR assays. To better define the involvement of F. necrophorum in the aetiology of these lesions, a qPCR targeting the rpoB gene was employed and confirmed the presence of F. necrophorum DNA in both the control and lesions swab samples, although the mean F. necrophorum genome copy number detected in the lesion swab samples was ∼19-fold higher than detected in the contralateral control swab samples (245 versus 4752 genome copies/µl, respectively; P < 0.001). Although we have not been able to conclusively define an aetiological agent, the presence of both F. necrophorum and S. dysgalactiae in the majority of lesions assayed supports their role in the aetiopathogenesis of these lesions.

Putative ß-Barrel Outer Membrane Proteins of the Bovine Digital Dermatitis-Associated Treponemes: Identification, Functional Characterization, and Immunogenicity.

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative ß-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted ß-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as ß-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly ß-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal ß-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.