Bovine Digital Dermatitis Treponemes Research Articles

Survival of bovine digital dermatitis treponemes in conditions relevant to the host and farm environment

ObjectivesBovine digital dermatitis (BDD), a painful infectious foot disease in dairy cattle, endemic in many countries worldwide, causes substantial economic and welfare impacts. Treponema spp. are considered key to BDD pathogenesis. To aid infection reservoir identification and control measure development, survival of BDD treponemes was investigated in different temperatures (4, 12, 20, 37, 45 and 60 °C), pH values (5–9.0), dairy cattle faeces and bedding types: straw shavings, sand, sand containing 5% lime (w/w) and recycled manure solids (RMS). MethodsA turbidity microplate methodology was adapted to measure pH impact on growth. Survival of BDD treponemes for the different conditions were assessed by sub-cultures of microcosms over different time points. ResultsBDD treponemes remained viable between 4 and 37 °C and pH 5.5 and 9.0 under anaerobic conditions. In sterile faecal microcosms, incubated aerobically at 12 °C, BDD treponemes remained viable for a median of 1 day (15 min - 6 day range). Variation in duration of survival and ability to grow was observed between phylogroups and strains. In aerobic microcosms, T. phagedenis T320A remained viable for the full 7 days in sand, 6 days in sawdust, 5 days in RMS, but was not viable after 15 min in straw or sand containing 5% (w/w) lime. ConclusionsTreponeme survival conditions identified here should enhance future BDD infection reservoir surveys and enable control measures. Of note, straw or sand containing 5% (w/w) lime should be assessed in BDD field trials. Finally, these data indicate BDD treponemes exhibit characteristics of facultative anaerobes.

Dissecting the molecular diversity and commonality of bovine and human treponemes identifies key survival and adhesion mechanisms.

Here, we report the first complete genomes of three cultivable treponeme species from bovine digital dermatitis (DD) skin lesions, two comparative human treponemes, considered indistinguishable from bovine DD species, and a bovine gastrointestinal (GI) treponeme isolate. Key genomic differences between bovine and human treponemes implicate microbial mechanisms that enhance knowledge of how DD, a severe disease of ruminants, has emerged into a prolific, worldwide disease. Bovine DD treponemes have additional oxidative stress genes compared to nearest human-isolated relatives, suggesting better oxidative stress tolerance, and potentially explaining how bovine strains can colonize skin surfaces. Comparison of both bovine DD and GI treponemes as well as bovine pathogenic and human non-pathogenic saprophyte Treponema phagedenis strains indicates genes encoding a five-enzyme biosynthetic pathway for production of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, a rare di-N-acetylated mannuronic acid sugar, as important for pathogenesis. Bovine T. phagedenis strains further differed from human strains by having unique genetic clusters including components of a type IV secretion system and a phosphate utilisation system including phoU, a gene associated with osmotic stress survival. Proteomic analyses confirmed bovine derived T. phagedenis exhibits expression of PhoU but not the putative secretion system, whilst the novel mannuronic acid pathway was expressed in near entirety across the DD treponemes. Analysis of osmotic stress response in water identified a difference between bovine and human T. phagedenis with bovine strains exhibiting enhanced survival. This novel mechanism could enable a selective advantage, allowing environmental persistence and transmission of bovine T. phagedenis. Finally, we investigated putative outer membrane protein (OMP) ortholog families across the DD treponemes and identified several families as multi-specific adhesins capable of binding extra cellular matrix (ECM) components. One bovine pathogen specific adhesin ortholog family showed considerable serodiagnostic potential with the Treponema medium representative demonstrating considerable disease specificity (91.6%). This work has shed light on treponeme host adaptation and has identified candidate molecules for future diagnostics, vaccination and therapeutic intervention.

Challenge of Bovine Foot Skin Fibroblasts With Digital Dermatitis Treponemes Identifies Distinct Pathogenic Mechanisms.

Bovine digital dermatitis (BDD) is a common infectious disease of digital skin in cattle and an important cause of lameness worldwide, with limited treatment options. It is of increasing global concern for both animal welfare and food security, imposing a large economic burden on cattle farming industries each year. A polytreponemal etiology has been consistently identified, with three key phylogroups implicated globally: Treponema medium, Treponema phagedenis, and Treponema pedis. Pathogenic mechanisms which might enable targeted treatment/therapeutic development are poorly defined. This study used RNA sequencing to determine global differential mRNA expression in primary bovine foot skin fibroblasts following challenge with three representative BDD treponemes and a commensal treponeme, Treponema ruminis. A pro-inflammatory response was elicited by the BDD treponemes, mediated through IL-8/IL-17 signaling. Unexpectedly, the three BDD treponemes elicited distinct mechanisms of pathogenesis. T. phagedenis and T. pedis increased abundance of mRNA transcripts associated with apoptosis, while T. medium and T. pedis increased transcripts involved in actin rearrangement and loss of cell adhesion, likely promoting tissue invasion. The upregulation of antimicrobial peptide precursor, DEFB123, by T. phagedenis spirochaetes may present a microbial ecological advantage to all treponemes within BDD infected tissue, explaining their dominance within lesions. A commensal, T. ruminis, significantly dysregulated over three times the number of host mRNA transcripts compared to BDD treponemes, implying BDD treponemes, akin to the syphilis pathogen (Treponema pallidum), have evolved as “stealth pathogens” which avoid triggering substantial host immune/inflammatory responses to enable persistence and tissue invasion. Immunohistochemistry demonstrated increased IL-6, IL-8, RND1, and CFB protein expression in BDD lesions, confirming in vitro fibroblast observations and highlighting the system’s value in modeling BDD pathogenesis. Several unique shared gene targets were identified, particularly RGS16, GRO1, MAFF, and ZC3H12A. The three key BDD Treponema phylogroups elicited both distinct and shared pathogenic mechanisms in bovine foot skin; upregulating inflammation whilst simultaneously suppressing adaptive immunity. The novel gene targets identified here should enable future vaccine/therapeutic approaches.

Genomic Detection of Bovine Digital Dermatitis Treponemes in Sole Ulcers

Objective- Evaluation of possible contamination of sole ulcers with digital dermatitis Treponema spp . Design- Clinical detection Animals- Thirty-three cows with sole ulcer lesion. Procedures- Hoof trimmings were done by professional veterinarian hoof trimmer on a regular basis. Sole ulcer and digital dermatitis recorded as a wounded lesion in zone 4 and 10 of the hooves during hoof trimming and recorded in special sheets. A total of Thirty-three tissue samples from sole ulcers in zone 4 of digits were taken and the 16S rRNA gene was followed by PCR assay. Results- Annual incidence of clinical digital dermatitis recorded as 7.1 percent as the second prevalent digital lesion in the farm following sole ulcer. Treponema spp . was found in 42 percent of the samples, none of the positive samples show concurrent lesion of digital dermatitis and sole ulcer.Conclusion and Clinical Relevance- Since no concurrent lesions were found in sole ulcer samples, finding Treponema spp. in tissues may be a result of the possible pathogenic presence of the bacteria in this region. Multifactorial causes of sole ulcer make different clinical presentations for the lesion and a microbial cause for the sole ulcer that led to non-healing ulcers were reported.

Survival of bovine digital dermatitis treponemes on hoof knife blades and the effects of various disinfectants

BackgroundBovine digital dermatitis (BDD) is a painful infectious foot disease of cattle, and much evidence implicates a pathogenic role for treponemes. This study measured the survival of BDD treponemes on...

High-level association of bovine digital dermatitis Treponema spp. with contagious ovine digital dermatitis lesions and presence of Fusobacterium necrophorum and Dichelobacter nodosus.

Contagious ovine digital dermatitis (CODD) is an important foot disease in sheep, with significant animal welfare and economic implications. It is thought that CODD emerged from bovine digital dermatitis (BDD) via treponemal bacteria. With wildlife species such as elk now suffering a CODD-like disease, it is imperative to clarify these disease etiologies. A large investigation into treponemal association with CODD is warranted. CODD lesions (n = 58) and healthy sheep foot tissues (n = 56) were analyzed by PCR for the three BDD-associated Treponema phylogroups and two other lameness-associated bacteria, Dichelobacter nodosus and Fusobacterium necrophorum. Spirochete culture was also attempted on CODD lesions. "Treponema medium/Treponema vincentii-like," "Treponema phagedenis-like," and Treponema pedis spirochetes were identified in 39/58 (67%), 49/58 (85%), and 41/58 (71%) of CODD lesions, respectively. One or more BDD-associated Treponema phylogroups were detected in 100% of CODD lesions. Healthy foot tissues did not amplify BDD-associated Treponema phylogroup DNA. D. nodosus and F. necrophorum were present in 34/58 (59%) and 41/58 (71%) of CODD lesions and 22/56 (39%) and 5/56 (9%) of healthy foot tissues, respectively. Thirty-two spirochetes were isolated from CODD lesions, with representatives clustering with, and indistinguishable from, each of the three BDD-associated Treponema phylogroups based on 16S rRNA gene comparisons. This study for the first time demonstrates a high-level association for BDD treponeme phylogroups in CODD and their absence from healthy tissues, supporting the hypothesis that BDD treponemes play a primary causative role in CODD and confirming that the specific PCR assays are an effective differential diagnostic tool for CODD.

Occurrence of Treponema DNA in equine hoof canker and normal hoof tissue.

Equine hoof canker is a chronic pododermatitis of still unknown aetiology. Recent findings reported for 3 canker-bearing individuals are suggestive for Treponema spp. having a role in disease pathogenesis. Based on this hypothesised association, we assessed a larger number of DNA samples from hooves with canker and normal hooves for the presence of treponemal DNA. Retrospective survey of archived material. The study involved 71 archival, PCR-compatible DNA extractions purified from 59 canker samples obtained from 26 equine cases and from 12 hoof biopsies taken from 9 canker-free control horses. Presence of treponemal DNA was assessed by qualitative PCR using 4 different primer pairs recognising in sum a broad range of Treponema ssp. Obtained amplification products were identified by bidirectional sequencing and BLAST alignment. Treponemal DNA was detected in 37 of 59 canker DNA samples from 19 of 26 cases and in 9 of 12 hoof DNA samples from 7 of 9 healthy individuals. Canine oral Treponema sp. and Treponema medium ssp. bovis were the most frequently detected treponemal sequences in hoof canker, while control tissues were mainly shown to harbour Treponema refringens-like or canine oral Treponema-like DNA. All control samples tested negative for T. medium ssp. bovis DNA. Treponema DNA was detectable in the majority of hoof canker and control samples. The sample groups differed to some extent regarding identified Treponema phylotypes; however, this finding may be explained by the methodology used. Treponemes that are highly similar to bovine digital dermatitis treponemes are present in canker lesions. However, further work is needed to clarify the specific contribution of the identified Treponema phylotypes to the pathogenesis of disease.

Differential inflammatory responses of bovine foot skin fibroblasts and keratinocytes to digital dermatitis treponemes

Bovine digital dermatitis (BDD) is a serious infectious inflammatory lameness causing pain and suffering to many cattle worldwide and which has severe economic implications. This study set out to investigate relationships between the treponemes considered causal of BDD and the local inflammatory response of the bovine host. Here we describe, for the first time, the isolation of bovine foot skin keratinocytes and fibroblasts as separate cell lineages. These cell lines were then exposed to treponeme whole-cell sonicates, and the gene expression of selected host inflammatory mediators investigated using quantitative reverse transcriptase PCR. Several genes, including those encoding RANTES/CCL5, MMP12, TNFα, TGFβ and TIMP3 were significantly upregulated in fibroblasts exposed to whole-cell sonicates derived from BDD treponeme phylotypes. For each of the above genes there were similar fibroblast expression increases for all three BDD treponeme phylotypes tested, suggesting common virulence mechanisms. With bovine foot skin keratinocytes, we were unable to detect expression of RANTES/CCL5 and after incubation with BDD treponeme constituents we were unable to observe any significant changes in expression of inflammatory mediators tested. These contrasting results suggest fibroblasts rather than keratinocytes may be an important shared target of pathogenesis for BDD treponemes.

Host and environmental reservoirs of infection for bovine digital dermatitis treponemes

Bovine digital dermatitis (BDD) is a global infectious disease causing lameness of cattle and is responsible for substantial animal welfare issues and economic losses. The causative agents are considered to be spirochetal bacteria belonging to the genus Treponema, which have consistently been identified in BDD lesions worldwide. One potential means of controlling infection is the disruption of transmission; however, the infection reservoirs and transmission routes of BDD treponemes have yet to be elucidated. To address these issues, we surveyed for evidence of BDD treponeme presence in the dairy farm environment, in bovine tissues and in bovine gastrointestinal (GI) tract contents. A total of 368 samples were tested using PCR assays specific for each of three currently recognised, isolated phylotypes of BDD treponemes. All environmental samples, together with insects and GI tract content samples were negative for BDD treponeme DNA from the three phylotypes. However, we identified BDD treponemes in two non-pedal bovine regions: the oral cavity (14.3% of cattle tested) and the rectum (14.8% of cattle tested). Whilst only single phylotypes were detected in the oral cavity, two of the rectal tissues yielded DNA from more than one phylotype, with one sample yielding all three BDD treponeme phylotypes. Whilst it might be considered that direct skin to skin contact may be a major transmission route of BDD treponemes, further studies are required to characterise and determine the potential contribution of oral and rectal carriage to BDD transmission.

Association between bovine digital dermatitis treponemes and a range of ‘non-healing’ bovine hoof disorders

This study describes the association between bovine digital dermatitis (BDD) treponemes and three ‘non-healing’ bovine hoof horn lesions, namely, ‘toe necrosis’ (TN), ‘non-healing white line disease’ (nhWLD) and ‘non-healing sole...

Understanding bovine digital dermatitis

David Logue assesses the significance of a paper summarised in this week's Veterinary Record describing an association between bovine digital dermatitis treponemes and three ‘non-healing’ bovine hoof horn lesions BOVINE digital dermatitis was first described in Italy by [Cheli and Mortellaro (

Characterization of Novel Bovine Gastrointestinal Tract Treponema Isolates and Comparison with Bovine Digital Dermatitis Treponemes

This study aimed to isolate and characterize treponemes present in the bovine gastrointestinal (GI) tract and compare them with bovine digital dermatitis (BDD) treponemes. Seven spirochete isolates were obtained from the bovine GI tract, which, on the basis of 16S rRNA gene comparisons, clustered within the genus Treponema as four novel phylotypes. One phylotype was isolated from several different GI tract regions, including the omasum, colon, rumen, and rectum. These four phylotypes could be divided into two phylotype pairs that clustered closest with each other and then with different, previously reported rumen treponemes. The treponemes displayed great genotypic and phenotypic diversity between phylotypes and differed considerably from named treponeme species and those recently reported by metagenomic studies of the bovine GI tract. Phylogenetic inference, based on comparisons of 16S rRNA sequences from only bovine treponemes, suggested a marked divergence between two important groups. The dendrogram formed two major clusters, with one cluster containing GI tract treponemes and the other containing BDD treponemes. This division among the bovine treponemes is likely the result of adaptation to different niches. To further differentiate the bovine GI and BDD strains, we designed a degenerate PCR for a gene encoding a putative virulence factor, tlyC, which gave a positive reaction only for treponemes from the BDD cluster.

Association of Unique, Isolated Treponemes with Bovine Digital Dermatitis Lesions

This study used a PCR-based approach targeting 16S rRNA gene fragments to determine the occurrence and association of the three bovine digital dermatitis (BDD) treponeme phylogroups within lesions found in cattle from the United Kingdom. Examination of 51 BDD lesions collected from infected cattle across the United Kingdom revealed that BDD treponeme group 1 (Treponema medium/Treponema vincentii-like), group 2 (Treponema phagedenis-like), and group 3 (Treponema putidum/Treponema denticola-like) were present in 96.1%, 98%, and 76.5% of BDD lesions, respectively. The three phylogroups were present together in 74.5% of lesions. The PCR assays enabled the isolation of further treponeme strains from previously mixed primary BDD lesion cultures. Here a representative from each of the three distinct treponeme phylogroups was isolated from a single BDD lesion for the first time. These data highlight the extent to which this disease is polytreponemal. Immunohistochemistry and electron microscopy were used to investigate lesional hoof tissues, resulting in treponemes being identified copiously in hair follicles and sebaceous glands, suggesting a potential route of exit and/or entry for these pathogens. This study gives further evidence for the importance of the three treponeme groups in BDD pathogenesis and reiterates the value of molecular genetic approaches for isolating and identifying fastidious anaerobes.