The growth-stimulating factor of Treponema phagedenis from bovine digital dermatitis lesions.

Bovine digital dermatitis (BDD) is an infectious skin disease of the hoof characterized by painful ulcerations that cause lameness in dairy cattle. Cell-free supernatants (CFS) of Falsiporphyromonas endometrii predominantly isolated from BDD lesions had the highest growth-stimulating effect on Treponema phagedenis among BDD-associated bacteria. Butyric acid was detected at a concentration of 45.4 mM in CFS of F. endometrii, and the growth of T. phagedenis was significantly promoted by butyric acid supplementation.

Adherence and metal-ion acquisition gene expression increases during infection with Treponema phagedenis strains from bovine digital dermatitis.

Digital dermatitis (DD) is an ulcerative foot lesion on the heel bulbs of dairy cattle. DD is a polymicrobial disease with no precise etiology, although Treponema spirochetes are found disproportionally abundant in diseased tissue. Within Treponema, several different species are found in DD; however, the species Treponema phagedenis is uniformly found in copious quantities and deep within the skin layers of the active, ulcerative stages of disease. The pathogenic mechanisms these bacteria use to persist in the skin and the precise role they play in the pathology of DD are widely unknown. To explore the pathogenesis and virulence of Treponema phagedenis, newly isolated strains of this species were investigated in a subcutaneous murine abscess model. In the first trial, a dosage study was conducted to compare the pathogenicity of different strains across three different treponemes per inoculum (TPI) doses based on abscess volumes. In the second trial, the expression levels of 11 putative virulence genes were obtained to gain insight into their involvement in pathogenesis. During the RT-qPCR analysis, it was determined that genes encoding for two metal-ion import lipoproteins and two adherence genes were found highly upregulated during infection. Conversely, two genes involved in motility and chemotaxis were found to not be significantly upregulated or utilized during infection. These results were supported by gene expression data from natural M2 lesions of dairy cattle. This gene expression analysis could highlight the preference in strategy for T. phagedenis to persist and adhere in the host rather than engage in motility and disseminate.

Digital dermatitis in Swedish dairy herds assessed by ELISA targeting Treponema phagedenis in bulk tank milk.

BACKGROUND: Digital dermatitis (DD) is a contagious hoof infection affecting cattle worldwide. The disease causes lameness and a reduction in animal welfare, which ultimately leads to major decreases in milk production in dairy cattle. The disease is most likely of polymicrobial origin with Treponema phagedenis and other Treponema spp. playing a key role; however, the etiology is not fully understood. Diagnosis of the disease is based on visual assessment of the feet by trained hoof-trimmers and veterinarians, as a more reliable diagnostic method is lacking. The aim of this study was to evaluate the use of an enzyme-linked immunosorbent assay (ELISA) on bulk tank milk samples testing for the presence of T. phagedenis antibodies as a proxy to assess herd prevalence of DD in Swedish dairy cattle herds. RESULTS: Bulk tank milk samples were collected in 2013 from 612 dairy herds spread across Sweden. A nationwide DD apparent prevalence of 11.9% (8.1-14.4% CI95%) was found, with the highest proportion of test-positive herds in the South Swedish regions (31.3%; 19.9-42.4% CI95%). CONCLUSIONS: This study reveals an underestimation of DD prevalence based on test results compared to hoof trimming data, highlighting the critical need for a reliable and accurate diagnostic method. Such a method is essential for disease monitoring and the development of effective control strategies. The novelty of ELISA-based diagnostic methods for DD, coupled with the disease's polymicrobial origin, suggests an avenue for improvement. Developing an expanded ELISA, incorporating antigens from various bacterial species implicated in the disease, could enhance diagnostic accuracy. The significance of this study is underscored by the extensive analysis of a substantial sample size (612). Notably, this investigation stands as the largest assessment to date, evaluating the application of ELISA on bulk tank milk for DD diagnosis at the herd level.

Molecular Diagnosis of Footrot and Contagious Ovine Digital Dermatitis in Small Ruminants in the Iberian Peninsula.

Contagious ovine digital dermatitis (CODD) and footrot (FR), a sub-acute or acute necrotic (decaying) infectious disease involving the hoof and underlying tissues, pose economic challenges to herds in Spain and worldwide. The aetiological agent for FR is Dichelobacter nodosus, while CODD is caused by pathogenic Treponema phylogroups. We detail the findings derived from the analysis by qPCR of 105 pooled samples from 100 ovine and five caprine herds in Spain and Portugal, alongside 15 samples from healthy flocks in order to identify Dichelobacter nodosus, Fusobacterium necrophorum, Treponema spp., and three pathogenic Treponema phylogroups (T. phagedenis, T. medium, and T. pedis). Treponema spp. were detected in all 120 pools, including samples from the 15 healthy flocks where only one positive result for F. necrophorum was recorded. Mixed infections by agents different from Treponema spp. were identified in 68.57% of samples. Positive results for F. necrophorum and/or D. nodosus, were obtained for 91.4% of the pools, whereas the presence of the three pathogenic Treponema phylogroups was rare: each of them appeared in isolation in a single pool, while they were found in 18 pools in combination with other agents. While F. necrophorum was the sole finding in 16.2% of samples from affected herds, D. nodosus (the footrot causative agent) was only detected in 61% of affected farms. An improved qPCR protocol was implemented to determine the serogroups of D. nodosus in the samples and found all of them (except the G serogroup), often in combined infections (35.1%). This report concludes with comprehensive proposals for diagnosing, preventing, and treating hoof ailments, remarking the interest of the information about D. nodosus serogroups in order to improve the efficiency of immunization by choosing appropriate vaccine protocols.

Case Report: Detection of Treponema phagedenis in cerebrospinal fluid of a neurosyphilis patient by metagenomic next-generation sequencing.

Treponema phagedenis, a human commensal spirochete, has been reported world-wide as a key factor in the pathogenesis of bovine digital dermatitis. Here we report a case of T. phagedenis sequence detection in the cerebrospinal fluid (CSF) of a patient. The patient was diagnosed with neurosyphilis, and T. phagedenis was detected as the only microorganism in his CSF by metagenomic sequencing. The patient went through a round of penicillin therapy previously (2.4 million units of Benzathine Penicillin intramuscularly once a week for three weeks) that did not resolve the symptoms; after the diagnosis of neurosyphilis he was treated with Penicillin G Sodium 4.0 million units q4h intravenous for 14 days then his symptoms resolved. To the best of our knowledge, T. phagedenis has never been reported to be detected in a human's CSF before. This was also the first time it was detected by metagenomic next-generation sequencing. We propose that more etiological tests should be performed including culture and sequencing for more patients with syphilis, which will contribute to a deeper understanding of the pathogenicity of the spirochete.

Contagious Ovine Digital Dermatitis: A Novel Bacterial Etiology and Lesion Pathogenesis.

Contagious ovine digital dermatitis (CODD) is a severe and common infectious foot disease of sheep and a significant animal welfare issue for the sheep industry in the UK and some European countries. The etiology and pathogenesis of the disease are incompletely understood. In this longitudinal, experimental study, CODD was induced in 18 sheep, and for the first time, the clinical lesion development and associated microbiological changes in CODD affected feet are described over time, resulting in a completely new understanding of the etiopathogenesis of CODD. The majority of CODD lesions (83.9%) arose from pre-existing interdigital dermatitis (ID) and/or footrot (FR) lesions. All stages of foot disease were associated with high levels of poly-bacterial colonization with five pathogens, which were detected by quantitative PCR (qPCR): Treponema medium, Treponema phagedenis, Treponema pedis, Dichelobacter nodosus, and Fusobacterium necrophorum. Temporal colonization patterns showed a trend for early colonization by T. phagedenis, followed by F. necrophorum and D. nodosus, T. medium, and then T. pedis, D. nodosus was present at significantly higher predicted mean log10 genome copy numbers in FR lesions compared to both ID and CODD, while Treponema species were significantly higher in CODD and FR lesions compared to ID lesions (p < 0.001). Treatment of CODD-affected sheep with two doses of 10 mg/kg long acting amoxicillin resulted in a 91.7% clinical cure rate by 3 weeks post-treatment; however, a bacteriological cure was not established for all CODD-affected feet. The study found that in an infected flock, healthy feet, healed CODD feet, and treated CODD feet can be colonized by some or all of the five pathogens associated with CODD and therefore could be a source of continued infection in flocks. The study is an experimental study, and the findings require validation in field CODD cases. However, it does provide a new understanding of the etiopathogenesis of CODD and further supportive evidence for the importance of current advice on the control of CODD; namely, ensuring optimum flock control of footrot and prompt isolation and effective treatment of clinical cases.

Transcellular penetration of Treponema phagedenis isolated from papillomatous digital dermatitis in polarized normal human epidermal keratinocytes in vitro.

Papillomatous digital dermatitis (PDD) is a polymicrobial infection causing lameness in dairy cattle. Culture-independent analysis has shown that Treponema phagedenis is present consistently and predominantly in the lesions. However, the pathogenesis of PDD, especially the tissue penetration pathway, has not been examined. In the present study, we investigated whether T. phagedenis strains isolated from PDD produce proteolytic enzyme (s) for disruption of the epithelial cell barrier and have the ability to translocate in polarized normal human epidermal keratinocytes (NHEK) in vitro. Ten strains of T. phagedenis isolated from lesions did not show proteolytic activity on modified skim milk agar, although a human strain of T. denticola used as a control showed such activity. The integrity of tight junctions was monitored by measurement of transepithelial electrical resistance (TER). The TER values after inoculation of the T. phagedenis strains examined did not change during the experimental period; however, apical to basolateral translocation of T. phagedenis was confirmed after 24 hr by microscopy and Treponema-specific PCR. We further confirmed that translocation of T. phagedenis was accelerated by co-inoculation with live T. denticola, but not with heat-killed organisms. Furthermore, tight junction ZO-1 protein was not lost intensity after inoculation with T. phagedenis and the organism was observed in NHEK cells using a florescence microscope. These results suggest that T. phagedenis strains may translocate via a transcellular route in vitro and that the invasion is accelerated by other bacteria, such as T. denticola, producing proteolytic activity.

Treponema spp. Isolated from Bovine Digital Dermatitis Display Different Pathogenicity in a Murine Abscess Model.

Digital dermatitis (DD) causes lameness in cattle with substantial negative impact on sustainability and animal welfare. Although several species of Treponema bacteria have been isolated from various DD stages, their individual or synergistic roles in the initiation or development of lesions remain largely unknown. The objective of this study was to compare effects of the three most common Treponema species isolated from DD lesions in cattle (T. phagedenis, T. medium and T. pedis), both as individual and as mixed inoculations, in a murine abscess model. A total of 109 or 5 × 108Treponema spp. were inoculated subcutaneously, and produced abscess was studied after 7 days post infection. There were no synergistic effects when two or three species were inoculated together; however, T. medium produced the largest abscesses, whereas those produced by T. phagedenis were the smallest and least severe. Treponema species were cultured from skin lesions at 7 days post infection and, additionally, from the kidneys of some mice (2/5), confirming systemic infection may occur. Taken together, these findings suggest that T. medium and T. pedis may have more important roles in DD lesion initiation and development than T. phagedenis.

Genotypic and Phenotypic Characterization of Treponema phagedenis from Bovine Digital Dermatitis.

This study aimed to isolate and characterize Treponema spp. from bovine digital dermatitis (BDD)-infected dairy cattle. Seven isolates were characterized in this study. Isolates exhibited slow growth, and colonies penetrated the agar and exhibited weak ß-hemolysis. Round bodies were observed in old and antibiotic-treated cultures. Cells ranged from 9-12 µm in length, 0.2-2.5 µm in width, and were moderately spiraled. The 16S rRNA analysis revealed the isolates as Treponema phagedenis with >99% sequence homology. Isolates had alkaline phosphatase, acid phosphatase, ß-galactosidase, N-acetyl-ß-glucosaminidase, esterase (C4), esterase lipase (C8), naphthol-AS-BI-phosphohydrolase, and ß-glucuronidase activities. Low concentrations of ampicillin, erythromycin, and tetracycline were required to inhibit the growth of isolates. Formic, acetic, and butyric acids were produced, while propionic acid was significantly utilized, indicating its essentiality for treponemal growth. The isolates shared the same characteristics and, therefore, were considered as a single strain. Isolate HNL4 was deposited as a representative isolate (Treponema phagedenis KS1). The average nucleotide identity of strain KS1 showed a small difference with the human strain (99.14%) compared with bovine strain (99.72%). This study was the first to isolate and characterize Treponema phagedenis from BDD in Korea and, hence, it delivered pathogenicity-related insights and provided valuable information that can be used for the management of BDD.

High quality genome sequence of Treponema phagedenis KS1 isolated from bovine digital dermatitis.

Treponema phagedenis KS1, a fastidious anaerobe, was isolated from a bovine digital dermatitis (BDD)-infected dairy cattle in Chungnam, Korea. Initial data indicated that T. phagedenis KS1 exhibited putative virulent phenotypic characteristics. This study reports the whole genome assembly and annotation of T. phagedenis KS1 (KCTC14157BP) to assist in the identification of putative pathogenicity related factors. The whole genome of T. phagedenis KS1 was sequenced using PacBio RSII and Illumina HiSeqXTen platforms. The assembled T. phagedenis KS1 genome comprises 16 contigs with a total size of 3,769,422 bp and an overall guanine-cytosine (GC) content of 40.03%. Annotation revealed 3,460 protein-coding genes, as well as 49 transfer RNA- and 6 ribosomal RNA-coding genes. The results of this study provide insight into the pathogenicity of T. phagedenis KS1.