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.

Multilocus sequence typing of pathogenic treponemes isolated from contagious ovine digital dermatitis stage five lesions: Implications for disease transmission dynamics.

Contagious ovine digital dermatitis (CODD) causes a severe, infectious foot disease and lameness of sheep, is common within the UK and is now also emerging in other countries. As well as causing severe animal welfare issues, huge economic losses emerge from the disease due to weight loss/lack of weight gain, and veterinary treatments. CODD lesion progress is measured, with a scoring system from 1 (early lesions) to 5 (healed). Here, using samples from an experimental flock infected by natural means, samples were taken from CODD stage 5 lesions, post treatment, and subjected to bacterial isolation and MLST using previously published methods. Sequences were compared to others from the same flock, and those from previous studies. All CODD 5 lesions produced viable Treponema spp. bacteria. High levels of variation of bacteria were seen, with 12 sequence types (STs) for T. medium phylogroup (11 new), 15 STs for T. phagedenis phylogroup (9 new) and six T. pedis STs, of which two were new. This study shows that CODD stage 5 lesions still contain viable bacteria, representing all three known pathogenic Treponema spp. phylogroups, and these may thus play a role in disease transmission and epidemiology despite appearing healed after treatment. The high level CODD treponeme variability within an infected flock where sheep were bought from different sources, as might occur in common agricultural practice, may suggest reasons as to why the bacterial disease is difficult to treat, control and eradicate, and adds further complexity to the polybacterial pathogenesis of these lesions.

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.

A survey of foot disinfection practices for control of bovine digital dermatitis: Evaluating solution depth, footbath hygiene, and the potential of footbaths as infection reservoirs for Treponema species.

Bovine digital dermatitis remains a widespread endemic disease of dairy cattle worldwide. Footbathing is commonly used as a control measure and has significant economic and environmental impacts. Few studies document footbathing practices on dairy farms or evaluate their suitability for achieving foot disinfection. This study describes footbathing practices on 32 farms observed in the United Kingdom, Ireland, and the Netherlands. We measured solution depth throughout footbathing and observed levels below 7 cm on 9 out of 32 farms, which leads to inadequate foot coverage. Solution depth was associated with the number of cow passages and decreased by 1.2 cm for every 100 cow passages. We also describe levels of OM content (g/L) throughout footbathing as a proxy for footbath hygiene. Our data indicates that almost half of footbaths (15/32) became contaminated above the 20 g/L threshold to which veterinary biocides are tested for efficacy, and that OM content is associated with the number of cow passages per liter of footbathing solution provided. A multivariable mixed model predicted that 1 L of footbathing solution per cow should be sufficient to prevent excess contamination. As a further measure of hygiene, we tested a subset of footbath samples to quantify the amount of DNA present from the Treponema species which are considered instrumental in the etiology of digital dermatitis. We did not detect Treponema DNA in footbath samples, which suggested they are unlikely to act as infection reservoirs for this disease. Multivariable mixed models including farm identity as a random effect demonstrated that for both change in solution depth and OM content the effect of farm-level factors was large. Because of the magnitude of this farm effect, applying model predictions will not translate to adequate solution depth and hygiene on all farms. Our data highlights the importance of footbath auditing on individual farms.

Bovine Digital Dermatitis: Treponema spp. on trimming equipment and chutes - effect of washing and disinfection.

BACKGROUND: Digital dermatitis (DD) is a contagious bovine foot disease causing reduced animal welfare and negative economic consequences for the farmer. Treponema spp. are the most important causative agents. Studies indicate that trimming equipment can transfer DD-associated treponemes between cows. The aim of this observational study in 22 DD-positive Norwegian dairy herds was to investigate the risk of transferring Treponema spp. with trimming equipment and chutes after claw trimming, and after washing and disinfection. Swabs from the trimming equipment and chutes were collected from nine different locations, at five different time points. Bacterial DNA was extracted from 647 swabs and analysed by qPCR for Treponema spp. In addition, 172 swabs taken immediately after trimming, were analysed by a multiplex qPCR targeting T. phagedenis, T. pedis and T. medium/vincentii. Biopsy sampling from DD lesions was performed on cows in the same herds during trimming. Altogether 109 biopsies were analysed by FISH for confirmation of the DD diagnosis and identification of Treponema phylotypes (PTs). RESULTS: High numbers of Treponema spp. were detected from all nine locations on the trimming equipment and chutes immediately after trimming, and T. phagedenis was detected on two or more locations in all but two herds, 1 and 19. There was a decline in the amount of Treponema spp. after washing and disinfection. The belly belt, the cuff, and the footrest on the chute had the highest proportion of positive samples after disinfection. The belly belt had the highest copy numbers of all nine locations (median = 7.9, max = 545.1). No Treponema spp. was detected on the hoof knives after disinfection. Treponema phagedenis, T. pedis, and Treponema phylotype 3 (T. refringens) were detected by FISH analysis of the biopsies. Treponema phagedenis was detected in biopsies from all herds except 1 and 19. CONCLUSION: This study shows that DD-associated Treponema spp. were present on the trimming equipment and chutes after trimming cows in DD-positive herds. Washing and disinfection reduced the load of Treponema spp. However, large differences in Treponema spp. between different locations were documented. High copy numbers on the grinder and the chute after disinfection, indicates that sufficient cleaning and disinfection of these locations is difficult, and that passive transfer of DD-associated treponemes (viable or not) is possible.

Discovery of novel treponemes associated with pododermatitis in elk (Cervus canadensis).

Pododermatitis, also known as treponeme-associated hoof disease (TAHD), presents a significant challenge to elk (Cervus canadensis) populations in the northwestern USA, with Treponema spp. consistently implicated in the lesion development. However, identifying species-specific Treponema strains from these lesions is hindered by its culture recalcitrance and limited genomic information. This study utilized shotgun sequencing, in silico genome reconstruction, and comparative genomics as a culture-independent approach to identify metagenome-assembled Treponema genomes (MATGs) from skin scraping samples collected from captive elk experimentally challenged with TAHD. The genomic analysis revealed 10 new MATGs, with 6 representing novel genomospecies associated with pododermatitis in elk and 4 corresponding to previously identified species-Treponema pedis and Treponema phagedenis. Importantly, genomic signatures of novel genomospecies identified in this study were consistently detected in biopsy samples of free-ranging elk diagnosed with TAHD, indicating a potential etiologic association. Comparative metabolic profiling of the MATGs against other Treponema genomes showed a distinct metabolic profile, suggesting potential host adaptation or geographic uniqueness of these newly identified genomospecies. The discovery of novel Treponema genomospecies enhances our understanding of the pathogenesis of pododermatitis and lays the foundation for the development of improved molecular surveillance tools to monitor and manage the disease in free-ranging elk.IMPORTANCETreponema spp. play an important role in the development of pododermatitis in free-ranging elk; however, the species-specific detection of Treponema from pododermatitis lesions is challenging due to culture recalcitrance and limited genomic information. The study utilized shotgun sequencing and in silico genome reconstruction to identify novel Treponema genomospecies from elk with pododermatitis. The discovery of the novel Treponema species opens new avenues to develop molecular diagnostic and epidemiologic tools for the surveillance of pododermatitis in elk. These findings significantly enhance our understanding of the genomic landscape of the Treponemataceae consortium while offering valuable insights into the etiology and pathogenesis of emerging pododermatitis in elk populations.

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.

Digital dermatitis-associated Treponema species detection and quantification in migratory tundra caribou (Rangifer tarandus).

Treponema spp. are associated with infectious lameness in livestock and wild ruminants. While extensive research has been conducted on cattle, investigations in wild ruminants are scarce. Hoof disease is common in caribou populations (Rangifer tarandus), but investigations are limited due to the remoteness of the Arctic. Our study aimed to assess the presence of Treponema spp. associated with bovine digital dermatitis in caribou. DNA was extracted from coronary band tissues from forty-eight caribou without visible hoof lesions and analyzed using two PCR methods (qPCR and nPCR). Treponema spp. were detected in low copy numbers/mg of tissue (3.6 to 6.6 × 101). T. phagedenis was the most prevalent and abundant species in 58% of samples by qPCR, followed by T. medium (44%), and T. pedis (10%). The qPCR and nPCR agreement ranged between 65% and 75% (Cohen's kappa 0.22-0.51). Sanger sequencing of thirteen nPCR products confirmed that treponemes in caribou are remarkably similar to those found in domestic ruminants and wild elk. Our study highlights the colonization of treponemes in healthy hooves of a wild ruminant in the Arctic, where there is no presence of livestock, and expands knowledge on the host range and distribution of treponemes. These findings also emphasize the need for further research into the multifactorial nature of treponema-associated hoof diseases and the putative role of treponemes in infectious lameness affecting caribou.

Proinflammatory CD14highCD16low monocytes/macrophages prevail in Treponema phagedenis-associated bovine digital dermatitis.

Digital dermatitis (DD) is a skin disease in cattle characterized by painful inflammatory ulcerative lesions in the feet, mostly associated with local colonization by Treponema spp., including Treponema phagedenis. The reason why most DD lesions remain actively inflamed and progress to chronic conditions despite antibiotic treatment remains unknown. Herein, we show an abundant infiltration of proinflammatory (CD14highCD16low) monocytes/macrophages in active DD lesions, a skin response that was not mitigated by topical treatment with oxytetracycline. The associated bacterium, T. phagedenis, isolated from DD lesions in cattle, when injected subcutaneously into mice, induced abscesses with a local recruitment of Ly6G+ neutrophils and proinflammatory (Ly6ChighCCR2+) monocytes/macrophages, which appeared at infection onset (4 days post challenge) and persisted for at least 7 days post challenge. When exploring the ability of macrophages to regulate inflammation, we showed that bovine blood-derived macrophages challenged with live T. phagedenis or its structural components secreted IL-1ß via a mechanism dependent on the NLRP3 inflammasome. This study shows that proinflammatory characteristics of monocytes/macrophages and neutrophils dominate active non-healing ulcerative lesions in active DD, thus likely impeding wound healing after antibiotic treatment.

An in-depth investigation of the microbiota and its virulence factors associated with severe udder cleft dermatitis lesions.

Udder cleft dermatitis (UCD) is a skin condition affecting the anterior parts of the udder in dairy cattle. In the present study, we aimed to shed light on the microbiota in severe UCD lesions versus healthy udder skin by putting forward a taxonomic and functional profile based on a virulence factor analysis. Through shotgun metagenomic sequencing, we found a high proportion of bacteria in addition to a low abundance of archaea. A distinct clustering of healthy udder skin versus UCD lesion samples was shown by applying principal component analysis and (sparse) partial least squares analysis on the metagenomic data. Proteobacteria, Bacillota, and Actinomycetota were among the most abundant phyla in healthy udder skin samples. In UCD samples, Bacteroidota was the most abundant phylum. At genus level, Bifidobacterium spp. had the highest relative abundance in healthy skin samples, whereas Porphyromonas spp. and Corynebacterium spp. had the highest relative abundance in UCD samples. In the differential abundance analysis, Porphyromonas spp. and Bacteroides spp. were significantly differentially abundant in UCD samples, whereas Bifidobacterium spp., Staphylococcus sp. AntiMn-1, and Staphylococcus equorum were more commonly found in healthy samples. Moreover, the abundance of several treponeme phylotypes was significantly higher in lesion samples. The streptococcal cysteine protease speB was among the most abundant virulence factors present in severe UCD lesions, while a plethora of virulence factors such as the antitoxin relB were downregulated, possibly contributing to creating the ideal wound climate for the dysbiotic community. Network analysis showed healthy lesion samples had a large network ofpositive, correlations between the abundances of beneficial species such as Aerococcus urinaeequi and Bifidobacterium angulatum, indicating that the healthy skin microbiome forms an active protective bacterial network, which is disrupted in case of UCD. In UCD samples, a smaller microbial network mainly consisting of positive correlations between the abundances of Bacteroides fragilis and anaerobic Bacteroidota was exposed. Moreover, a high correlation between the taxonomic data and virulence factors was revealed, concurrently with 2 separate networks of microbes and virulence factors. One network, matching with the taxonomic findings in the healthy udder skin samples, showcased a community of harmless or beneficial bacteria, such as Bifidobacterium spp. and Butyrivibrio proteoclasticus, associated with hcnB, hcnC, relB, glyoxalase, and cupin 2. The other network, corresponding to UCD samples, consisted of pathogenic or facultative pathogenic and mainly anaerobic bacteria such as Treponema spp., Mycoplasmopsis spp., and bovine gammaherpesvirus 4, that correlated with virulence factors SpvB, fhaB, and haemagglutination activity domain-associated factor. Our results point toward a dysbiotic community with a notable decrease in diversity and evenness, with a loss of normal skin inhabitants and innocuous or useful species making way for predominantly anaerobic, facultative pathogens. The shift in the abundance of virulence factors such as fhaB and SpvB could play a role in the manifestation of a local micro-environment favorable to the microbiome associated with udder skin lesions. Lastly, the presence of specific networks between microbial species, and between microbes and virulence factors was shown.

Quantifying and mapping digital dermatitis-associated bacteria in lesion and nonlesion body sites and dairy farm environment.

The source of infection of digital dermatitis (DD), an infectious lameness condition, is still uncertain. In this cross-sectional study, we aimed to identify potential reservoirs of DD bacteria in dairy cattle body sites with different stages of the disease and farm environments. We collected skin swabs from 85 dairy cows from 5 herds, 3 with and 2 without DD, from foot, hock, and udder cleft skin (with lesions or not), saliva, urine, and feces. We also obtained environmental samples. Real-time quantitative PCR targeted Treponema phagedenis, Treponema medium, Treponema pedis, Porphyromonas levii, Bacteroides pyogenes, Fusobacterium necrophorum, and Fusobacterium mortiferum. Digital dermatitis-associated Treponema spp. were exclusively detected in DD-affected herds in DD-foot and other skin lesions, healthy skin, saliva, and environmental samples. In contrast, the non-Treponema spp. were found in samples from both DD-negative and affected herds. As expected, DD lesions had higher bacterial loads than healthy skin. Interestingly, similar counts were observed in udder cleft lesions, indicating a potential opportunistic behavior on compromised skin. None of the targeted species were detected in fecal samples, but P. levii, B. pyogenes, and F. necrophorum were detected in urine. All 7 species were detected in saliva, although in low quantities. No associations were observed between the presence of each bacterial species in DD lesions and urine; however, there was an association between the presence of DD-Treponema spp. in lesions and saliva, hock, and udder skin. Feces and urine do not seem to be a DD bacteria primary source, but saliva and other skin lesions may play a role. Longitudinal studies would improve our understanding of DD-associated bacteria's transient or persistent presence in these sites. Investigating the sources of DD-associated bacteria will guide future interventions to minimize bacterial shedding and transmission, ultimately more effectively reducing bacterial load, transmission, and sources of infection in dairy herds.

[Risk factors for chronic perforating skin lesions in the area of the digits in cattle on Swiss alpine pastures]. / Risikofaktoren für chronisch perforierende Hautläsionen im Zehenbereich beim Rind auf Schweizer Alpen.

INTRODUCTION: Diseases of the digits often occur in cattle on larger cattle mountain pastures. In the late spring 2020, at the time of the ascent of 1554 cattle to 11 high altitude alpine pastures in the Lower Engadine region, lesions in the area of the digits were clinically assessed and documented. 254 cattle were of non-cantonal and 1300 of local origin (Lower Engadine; postal code CH-75XX). Skin lesions in the area of the digits, identified as digital dermatitis (DD; Mortellaro's disease), were further classified according to the DD scoring system. Nonspecific skin lesions with clinical evidence of granulation tissue formation were termed chronic penetrating skin lesions (CPSL). At the end of the alpine pasturing season, in the early fall (descent of cattle from the alpine pastures), the procedure was repeated, and biopsies were taken from randomly selected cattle with CPSL. Digital dermatitis lesions were found in 34 of 1551 cattle at ascent, but no case of CPSL was found at that time. At descent, 19 of 1529 cattle had DD lesions and 88 cattle had CPSL. The clinical appearance of the CPSL was consistent with chronic skin lesions caused by penetrating skin lacerations. Histologically, the majority of the CPSL were classified as chronic hyperplastic dermatitis with granulation tissue formation. In all CPSL biopsies examined by PCR, Fusobacterium necrophorum and Porphyromonas levii, but neither Dichelobacter nodosus nor the tested Treponema species were detected. Fluorescence in situ hybridization revealed a negative result for Treponema species in all biopsies. In the regression analysis, cattle in the age group of 365 to 730 days had an increased risk for the presence of CPSL compared to the age group of 160 to 365 days (odds ratio (OR) = 4,95; confidence interval (CI) = 1,97-12,43). Holstein cattle had an increased risk of developing CPSL compared to Brown cattle (OR = 2,92; CI = 1,46-5,86) and cattle of non-cantonal origin showed a massively higher risk compared to local cattle (OR = 10,59; CI = 5,79 - 19,37). The statistically significant associations found in the present study can be taken into account in the selection of animals for summer pasturing on high altitudes in the future in order to reduce the prevalence of CPSL and consequently reduce the antimicrobial use. Spread of DD during the alpine pasturing season within the cattle groups examined was not found.

INTRODUCTION: Des atteintes aux onglons sont souvent observées sur les grands alpages de bovins. Des altérations au niveau des onglons ont été examinées cliniquement et répertoriées chez 1554 bovins lors de leur arrivée sur 11 alpages en Basse-Engadine, en provenance d'un autre canton (n = 254) ou de la localité à laquelle l'alpage appartenait (n = 1300, numéro postal 75XX), au moment de la montée à l'alpage en 2020. Les altérations cutanées diagnostiquées comme dermatite digitale (DD; maladie de Mortellaro) ont de plus été classifiées selon les scores en usage pour la DD. Les lésions cutanées non-spécifiques présentant une formation de tissu de granulation ont été enregistrées comme lésions cutanées perforantes chroniques (LCPC). La procédure a été répétée lors de la désalpe et une biopsie a été prise de chez des animaux présentant des LCPC choisis au hasard. Les caractéristiques de la topographie de l'alpage et celles du sol, ainsi que la densité d'occupation ont été enregistrées pour chaque alpage. Des lésions de DD ont été constatées chez 34 des 1551 bovins lors de la montée à l'alpage, mais aucun cas de LCPC n'a été observé. Lors de la désalpe, 19 des 1551 bovins présentaient des lésions de DD et 88 une LCPC. L'apparence des LCPC correspondait à des lésions cutanées chroniques après une blessure perforante de la peau. À l'histologie, il s'agissait la plupart du temps d'une dermatite chronique hyperplastique avec formation de tissu de granulation. Fusobacterium necrophorum et Porphyromonas levii ont été mis en évidence dans toutes les biopsies de LCPC soumises à une analyse par PCR, mais ni Dichelobacter nodosus ni les Treponema spp. recherchées n'ont été mis en évidence. L'hybridation in-situ en fluorescence était négative pour les tréponèmes dans toutes les biopsies. Selon les résultats d'une analyse de régression, les génisses âgées de 366 à 730 jours avaient un risque augmenté (Odds Ratio (OR) = 4,95; intervalle de confiance (IC) = 1,97 ­ 12,43) de présenter une LCPC en comparaison avec le groupe d'âge de 161 à 365 jours. Les bovins de race Holstein avaient un risque augmenté de présenter une LCPC en comparaison avec ceux de race grise (OR = 2,92; IC = 1,46 ­ 5,86), et les animaux en provenance d'autres cantons présentaient un risque massivement plus élevé que le cheptel local (OR = 10,59; IC = 5,79 ­ 19,37). Aucune différence significative n'a été observée dans la topographie ou dans la densité d'occupation entre les alpages avec et sans cas de LCPC. Les associations statistiquement significatives constatées dans cette étude peuvent être prises en compte à l'avenir lors de la sélection d'animaux pour l'alpage, dans le but de réduire la prévalence de LCPC, de diminuer la quantité d'antibiotiques administrés et d'améliorer le bien-être animal. Une propagation de la DD pendant la saison d'alpage n'a pas été constatée dans les groupes de bovins inclus dans l'étude.

Enhanced transformation efficiency in Treponema denticola enabled by SyngenicDNA-based plasmids lacking restriction-modification target motifs.

Oral spirochetes are among a small group of keystone pathogens contributing to dysregulation of tissue homeostatic processes that leads to breakdown of the tissue and bone supporting the teeth in periodontal disease. Additionally, our group has recently demonstrated that Treponema are among the dominant microbial genera detected intracellularly in tumor specimens from patients with oral squamous cell carcinoma. While over 60 species and phylotypes of oral Treponema have been detected, T. denticola is one of the few that can be grown in culture and the only one in which genetic manipulation is regularly performed. Thus, T. denticola is a key model organism for studying spirochete metabolic processes, interactions with other microbes, and host cell and tissue responses relevant to oral diseases, as well as venereal and nonvenereal treponematoses whose agents lack workable genetic systems. We previously demonstrated improved transformation efficiency using an Escherichia coli-T. denticola shuttle plasmid and its utility for expression in T. denticola of an exogenous fluorescent protein that is active under anaerobic conditions. Here, we expand on this work by characterizing T. denticola Type I and Type II restriction-modification (R-M) systems and designing a high-efficiency R-M-silent "SyngenicDNA" shuttle plasmid resistant to all T. denticola ATCC 35405 R-M systems. Resequencing of the ATCC 33520 genome revealed an additional Type I R-M system consistent with the relatively low transformation efficiency of the shuttle plasmid in this strain. Using SyngenicDNA approaches, we optimized shuttle plasmid transformation efficiency in T. denticola and used it to complement a defined T. denticola ΔfhbB mutant strain. We further report the first high-efficiency transposon mutagenesis of T. denticola using an R-M-silent, codon-optimized, himarC9 transposase-based plasmid. Thus, use of SyngenicDNA-based strategies and tools can enable further mechanistic examinations of T. denticola physiology and behavior.

Transmission and lesion progression of treponeme-associated hoof disease in captive elk (Cervus canadensis).

Treponeme-associated hoof disease (TAHD) is a debilitating disease of free-ranging elk (Cervus canadensis) in the northwestern U.S. While treponemes are associated with lesions, the etiology and transmissibility between elk are unknown. Our objective was to determine whether the disease can be environmentally transmitted to captive elk. Four individually housed treatment elk and 2 control elk were challenged with soil mixed with inoculum prepared from free-ranging elk hooves from TAHD-positive elk or autoclaved hooves from normal elk, respectively. The inoculum for each group was applied to the interdigital space and added to pre-existing soil in each pen. Eight challenges were conducted at 1-4-week intervals and lesion development was assessed during a 138-day challenge period that was followed by a 170-day monitoring period to document lesion progression. All treatment elk, but no control elk, developed gross and histologic lesions consistent with TAHD. Treponema phylotypes similar to those in bovine digital dermatitis in cattle were detected using 16S rRNA gene amplicon sequencing from lesions in all treatment elk, but no control elk, during the challenge period. Lesions progressed from ulcerations in the interdigital space to extensive ulceration and underrunning of the hoof capsule by 35 and 173 days following the initial inoculation, respectively. Lameness in treatment elk was correlated with lesion development (R = 0.702, p≤0.001), and activity of infected elk was reduced during the challenge (p≤0.001) and monitoring periods (p = 0.004). Body condition was significantly lower in treatment than control elk 168 days following the initial inoculation (p = 0.05) and at each individual elk's study endpoint (p = 0.006). Three of 4 treatment elk were euthanized when they reached humane endpoints, and one elk recovered. These results provide direct evidence that TAHD is a transmissible infectious disease in elk. As such, actions that reduce transmission risk can support disease management and prevention.

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

OBJECTIVES: Bovine 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). METHODS: A 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. RESULTS: BDD 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. CONCLUSIONS: Treponeme 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.

Evidence of novel Treponema phylotypes implicated in contagious ovine digital dermatitis and association of treponemes with major lameness causing foot pathogens.

In this study 269 swabs collected from 254 ovine foot lesions and 15 apparently healthy ovine feet were screened by PCR for the presence of major lameness causing foot pathogens viz. Treponema species, D. nodosus, F. necrophorum and T. pyogenes with the presumption that ovine foot lesion positive for Treponema species alone or in association with other three pathogens were categorized as contagious ovine digital dermatitis (CODD). While samples positive for D. nodosus alone or its combination with F. necrophorum and T. pyogenes were considered as footrot (FR) and samples in which F. necrophorum or T. pyogenes was found either alone or in combination were considered as interdigital dermatitis (ID). The overall occurrence of Treponema sp. in ovine foot lesions was 48.0%, and ranged from 33 to 58%. In Treponema positive samples D. nodosus, F. necrophorum and T. pyogenes were present in 34 (27.4%), 66 (54.4%) and 84 (68.5%) in contrast to Treponema negative samples in which these were present in 15 (11.1%), 20 (14.12%) and 17 (12.6%) samples, respectively. The data signifies that Treponema sp. are significantly associated with these foot pathogens and their different combinations with Treponema sp. influence the severity of CODD lesion. The identification of Treponema phylotypes was done by sequencing the 16S rRNA gene fragment of ten representative samples. Out of ten sequences, four (Trep-2, Trep-4, Trep-7 and Trep-10) were identical to Treponema sp. phylotype 1 (PT1) that belongs to phylogroup T. refringens-like, one sequence (Trep-1) was genetically close (90% sequence homology) to Treponema brennaborense while five sequences (Trep-3, Trep-5, Trep-6, Trep-8 and Trep-9) matched with uncultured bacterium clones of treponemes forming separate monophyletic group in phylogenetic tree and could represent new digital dermatitis phylogroup presently containing five ovine specific phylotypes. This is the first report on the presence of Treponema phylotypes other than three digital dermatitis (DD) Treponema phylogroups viz. T. phagedenis-like, T. medium/T. vincentii-like, and T. pedis-like that are frequently detected in CODD lesions. Metagenomic analysis of two representative samples revealed the abundance of genus Treponema in CODD lesion while this genus was absent in swab collected from clinically healthy foot suggesting that it might play primary role in producing CODD. These findings may further aid in understanding the etiopathogenesis of CODD and could help to develop appropriate treatment and mitigation strategies to combat the disease.

Development of a multiplex quantitative PCR assay for simultaneous detection of Treponema phagedenis, Treponema pedis, Treponema medium, and 'Treponema vincentii' and evaluation on bovine digital dermatitis biopsies.

Bovine digital dermatitis (BDD) is a contagious foot disease with worldwide occurrence in dairy cattle. The disease causes lameness and reduced animal welfare as well as economic losses for the farmer. The aetiology is not fully established but associations have been made with Treponema spp. Today, BDD diagnosis is mainly based on visual inspection of cattle feet, therefore this study aimed to develop a multiplex quantitative PCR (qPCR) assay targeting Treponema phagedenis, Treponema pedis, Treponema medium, and 'Treponema vincentii' to aid in diagnosis. The assay was tested for specificity on 53 bacterial strains and in silico on 168 Treponema spp. genomes, representative of at least 24 species. In addition, 37 BDD biopsies were analysed and the results compared to another qPCR assay published during the study period, which we modified by combining into a multiplex qPCR. The qPCR developed herein had a detection limit of 10 copies of each target species per PCR reaction. Both qPCR assays showed 100% specificity when tested on bacterial strains, but the qPCR developed in this study detected 3.4% more T. phagedenis-positive biopsies of lesion category M1-M4.1 than the modified assay. To conclude, the developed qPCR assay detecting T. phagedenis, T. pedis, T. medium, and 'T. vincentii' has high analytical sensitivity and specificity and provides a useful complementary tool for diagnosis and epidemiological studies of BDD. The assay could possibly also be used for contagious ovine digital dermatitis (CODD) as similar bacteriological profiles have been suggested for BDD and CODD, especially regarding certain Treponema spp.

Screening and diagnostic testing protocols for HIV and Syphilis infections in health care setting in Qatar: Evaluation and recommendations.

BACKGROUND: HIV and Syphilis are common STIs, which have become a concern and burden on healthcare systems, as many infections go untreated and lead to potentially serious complications. HIV is usually diagnosed with Western blot, PCR, and p24 antigen testing. Whereas, Syphilis is mainly diagnosed through clinical findings and serologic testing. The Medical Commission Department (MC) under MOPH is responsible for screening all newcomers to Qatar, aiming to keep the country free from serious infectious diseases. OBJECTIVE: We aimed to evaluate the diagnostic efficiency of the protocols used in the MC for screening HIV and Syphilis infections. METHODS: We conducted a retrospective study of samples analyzed by 4th Generation ARCHITECT® HIV Ag/Ab Combo and Rapid Plasma Reagin (RPR) between January to December 2019. ARCHITECT® HIV Ag/Ab Combo positive samples were confirmed by INNO-LIA™ HIVI/II and RT-PCR. RPR-reactive samples were confirmed by ARCHITECT® Syphilis Treponema pallidium Antibody (Syphilis TPA) assay. RESULTS: For HIV, data were collected from 585,587 individuals, of which 595 (0.1%) were positive by the ARCHITECT® HIV Ag/Ab Combo (Analyzer A). When all initially positive sera were re-tested on newly collected blood samples using different ARCHITECT® HIV Ag/Ab Combo analyzer (analyzer B), 99.8% (594/595) of samples were also positive, suggesting high reproducibility. The positive predictive value (PPV) between ARCHITECT® HIV Ag/Ab Combo and the INNO-LIA™ HIVI/II confirmatory assay was 31.8%. The PPV between ARCHITECT® HIV Ag/Ab Combo and HIV-PCR assay was 26.8%. Retrospective data for Syphilis were collected from a total of 97,298 individuals who visited the MC, of which 198 (0.20%) were initially positive by RPR. The PPV between RPR and Syphilis TPA confirmatory assay was 36.6%. CONCLUSION: Despite the high rate of false positivity using ARCHITECT® HIV Ag/Ab Combo and RPR screening assays, both assays have proven to be highly effective as screening testing methods.

Localization and pathogenic role of the cysteine protease dentipain in Treponema denticola.

The Msp protein complex and the serine protease dentilisin are the best-characterized virulence factors in Treponema denticola, the major etiological agent of chronic periodontitis. In addition to these outer sheath factors, the cysteine protease dentipain contributes to pathogenicity, but its secretion, processing, cellular localization, and role in T. denticola virulence are not fully understood. In this study, we found that full-sized dentipain (74-kDa) and the 52-kDa truncated form of the enzyme are located, respectively, in the outer sheath derived from T. denticola dentilisin- and the Msp-deficient mutants. Furthermore, dentipain was barely detected in the wild-type strain. These results suggest that dentilisin and Msp, the major outer sheath proteins, are involved in the secretion and maturation of dentipain. Inactivation of the dentipain gene slowed the growth of T. denticola, and the effect was more profound in serum-free medium than in serum-containing medium. Several genes, including those encoding transporters and methyl-accepting chemotaxis proteins, were differentially expressed in the dentipain-deficient mutant. Furthermore, the mutant strain was more hydrophobic than the wild-type strain. Finally, the mutant showed less autoaggregation activity and adhesion to IgG in a serum-free medium than the wild-type strain. These findings suggest that dentipain contributes to the virulence of T. denticola by facilitating adhesion and acquisition of nutrients essential for colonization and proliferation in the gingival crevice under serum-rich conditions.

Treponema spp. spirochetes and keratinopathogenic fungi isolated from keratomas in donkeys.

Keratoma is an aberrant keratin mass thought to originate from epidermal horn-producing cells interposed between the stratum medium of the hoof wall and the underlying third phalanx. The cause is unknown, although the presence of keratomas is frequently associated with chronic irritation, focal infection, or trauma. A total of 167 donkeys with keratomas were presented in this study. The diagnosis of a keratoma was based on clinical signs, radiography, and histopathologic examination. Surgical excision was attempted on all donkeys with lameness unless euthanasia was advised. Histopathologic examination, including Giemsa, periodic acid Schiff, and Young's silver special histochemical stains, was performed and showed the presence of fungal hyphae and spirochete bacteria within the degenerate keratin. Polymerase chain reaction (PCR) for treponeme bacteria was performed on 10 keratoma lesions and 9 healthy pieces of hoof (controls). All healthy donkey tissues were negative for the 3 recognized digital dermatitis (DD) treponeme phylogroups, whereas 3 of 10 (30%) donkey keratoma samples were positive for one of the DD treponeme phylogroups. Routine fungal culture and PCR for fungi were performed on 8 keratoma lesions and 8 healthy pieces of hoof (controls). Keratinopathogenic fungi were detected in 1 of 8 (12.5%) keratomas, while only non-keratinopathogenic, environmental fungi were detected in 8 control healthy hoof samples. This is the first time the DD treponemes phylogroup and keratinopathogenic fungi have been detected in keratomas. Further studies are required to assess the significance of this finding.