[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.

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.

The genomes of the yaws bacterium, Treponema pallidum subsp. pertenue, of nonhuman primate and human origin are not genomically distinct.

BACKGROUND: Treponema pallidum subsp. pertenue (TPE) is the causative agent of human yaws. Yaws is currently reported in 13 endemic countries in Africa, southern Asia, and the Pacific region. During the mid-20th century, a first yaws eradication effort resulted in a global 95% drop in yaws prevalence. The lack of continued surveillance has led to the resurgence of yaws. The disease was believed to have no animal reservoirs, which supported the development of a currently ongoing second yaws eradication campaign. Concomitantly, genetic evidence started to show that TPE strains naturally infect nonhuman primates (NHPs) in sub-Saharan Africa. In our current study we tested hypothesis that NHP- and human-infecting TPE strains differ in the previously unknown parts of the genomes. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we determined complete (finished) genomes of ten TPE isolates that originated from NHPs and compared them to TPE whole-genome sequences from human yaws patients. We performed an in-depth analysis of TPE genomes to determine if any consistent genomic differences are present between TPE genomes of human and NHP origin. We were able to resolve previously undetermined TPE chromosomal regions (sequencing gaps) that prevented us from making a conclusion regarding the sequence identity of TPE genomes from NHPs and humans. The comparison among finished genome sequences revealed no consistent differences between human and NHP TPE genomes. CONCLUSION/SIGNIFICANCE: Our data show that NHPs are infected with strains that are not only similar to the strains infecting humans but are genomically indistinguishable from them. Although interspecies transmission in NHPs is a rare event and evidence for current spillover events is missing, the existence of the yaws bacterium in NHPs is demonstrated. While the low risk of spillover supports the current yaws treatment campaign, it is of importance to continue yaws surveillance in areas where NHPs are naturally infected with TPE even if yaws is successfully eliminated in humans.

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.

Oral Spirochete Treponema denticola Intraoral Infection Reveals Unique miR-133a, miR-486, miR-126-3p, miR-126-5p miRNA Expression Kinetics during Periodontitis.

miRNAs are major regulators of eukaryotic gene expression and host immunity, and play an important role in the inflammation-mediated pathways in periodontal disease (PD) pathogenesis. Expanding our previous observation with the global miRNA profiling using partial human mouth microbes, and lack of in vivo studies involving oral spirochete Treponema denticola-induced miRNAs, this study was designed to delineate the global miRNA expression kinetics during progression of periodontitis in mice infected with T. denticola by using NanoString nCounter® miRNA panels. All of the T. denticola-infected male and female mice at 8 and 16 weeks demonstrated bacterial colonization (100%) on the gingival surface, and an increase in alveolar bone resorption (p < 0.0001). A total of 70 miRNAs with at least 1.0-fold differential expression/regulation (DE) (26 upregulated and 44 downregulated) were identified. nCounter miRNA expression profiling identified 13 upregulated miRNAs (e.g., miR-133a, miR-378) and 25 downregulated miRNAs (e.g., miR-375, miR-34b-5p) in T. denticola-infected mouse mandibles during 8 weeks of infection, whereas 13 upregulated miRNAs (e.g., miR-486, miR-126-5p) and 19 downregulated miRNAs (miR-2135, miR-142-3p) were observed during 16 weeks of infection. One miRNA (miR-126-5p) showed significant difference between 8 and 16 weeks of infection. Interestingly, miR-126-5p has been presented as a potential biomarker in patients with periodontitis and coronary artery disease. Among the upregulated miRNAs, miR-486, miR-126-3p, miR-126-5p, miR-378a-3p, miR-22-3p, miR-151a-3p, miR-423-5p, and miR-221 were reported in human gingival plaques and saliva samples from periodontitis and with diabetes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed various functional pathways of DE miRNAs, such as bacterial invasion of epithelial cells, Ras signaling, Fc gamma R-mediated phagocytosis, osteoclast differentiation, adherens signaling, and ubiquitin mediated proteolysis. This is the first study of DE miRNAs in mouse mandibles at different time-points of T. denticola infection; the combination of three specific miRNAs, miR-486, miR-126-3p, and miR-126-5p, may serve as an invasive biomarker of T. denticola in PD. These miRNAs may have a significant role in PD pathogenesis, and this research establishes a link between miRNA, periodontitis, and systemic diseases.

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.

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.

Longitudinal Variations in the tprK Gene of Treponema pallidum in an Amoy Strain-Infected Rabbit Model.

Heterogeneous tprK sequences have been hypothesized to be an important factor for persistent infection of Treponema pallidum subsp. pallidum (T. pallidum) in humans. Previous research has only explored tprK diversity using a rabbit model infected with almost clonal isolates, which is inconsistent with the fact that infected human isolates contain multiple heterogeneous tprK sequences. Here, we used the T. pallidum Amoy strain with heterogeneous tprK sequences to establish a rabbit infection model and explore longitudinal variations in the tprK gene under normal infection, immunosuppression treatment, and benzathine penicillin G (BPG) treatment using next-generation sequencing. The diversity of the tprK gene was high in all three groups but was highest in the control group and lowest in the BPG group. Interestingly, the overall diversity of tprK in all three groups decreased during infection, exhibiting a "more to less" trend, indicating that survival selection may be an important factor affecting tprK variation in the later infection stage. BPG treatment appeared to reduce the diversity of tprK but increased the frequency of predominant sequence changes, which might facilitate the escape of T. pallidum from the host immune clearance. Furthermore, the original predominant V region sequence did not disappear with disease progression but retained a relatively high proportion within the population, suggesting a new direction for tprK-related vaccine research. This study provides insights into longitudinal variations within the highly heterogeneous tprK gene sequences of T. pallidum and will contribute to further exploration of the pathogenesis of syphilis. IMPORTANCE The tprK variations are an important factor in persistent T. pallidum infection. A nearly clonal isolate has been used previously to investigate the mechanism of tprK gene variations; however, clinical T. pallidum isolates in infected humans exhibit multiple heterogeneous tprK sequences. Here, we use next-generation sequencing to explore longitudinal variations in the tprK gene under normal infection and immunosuppression and benzathine penicillin G treatment in a rabbit model infected with the Amoy strain with heterogeneous tprK sequences. The overall diversity of tprK in all three groups was high and decreased during infection, exhibiting a "more to less" trend. Benzathine penicillin G treatment reduced the diversity of tprK but increased the frequency of predominant sequence changes. Moreover, the original predominant V region sequence did not disappear as the disease progressed but remained at a relatively high proportion within the population. The research results give us a new understanding about tprK variation.

Biophysical and biochemical studies support TP0094 as a phosphotransacetylase in an acetogenic energy-conservation pathway in Treponema pallidum.

The mechanisms of energy generation and carbon-source utilization in the syphilis spirochete Treponema pallidum have remained enigmatic despite complete genomic sequence information. Whereas the bacterium harbors enzymes for glycolysis, the apparatus for more efficient use of glucose catabolites, namely the citric-acid cycle, is apparently not present. Yet, the organism's energy needs likely exceed the modest output from glycolysis alone. Recently, building on our structure-function studies of T. pallidum lipoproteins, we proposed a "flavin-centric" metabolic lifestyle for the organism that partially resolves this conundrum. As a part of the hypothesis, we have proposed that T. pallidum contains an acetogenic energy-conservation pathway that catabolizes D-lactate, yielding acetate, reducing equivalents for the generation and maintenance of chemiosmotic potential, and ATP. We already have confirmed the D-lactate dehydrogenase activity in T. pallidum necessary for this pathway to operate. In the current study, we focused on another enzyme ostensibly involved in treponemal acetogenesis, phosphotransacetylase (Pta). This enzyme is putatively identified as TP0094 and, in this study, we determined a high-resolution (1.95 Å) X-ray crystal structure of the protein, finding that its fold comports with other known Pta enzymes. Further studies on its solution behavior and enzyme activity confirmed that it has the properties of a Pta. These results are consistent with the proposed acetogenesis pathway in T. pallidum, and we propose that the protein be referred to henceforth as TpPta.

Clonal isolates of Treponema pallidum subsp. pallidum Nichols provide evidence for the occurrence of microevolution during experimental rabbit infection and in vitro culture.

The recent development of a system for long-term in vitro culture of the syphilis spirochete, Treponema pallidum subsp. pallidum, has introduced the possibility of detailed genetic analysis of this bacterium. In this study, the in vitro culture system was used to isolate and characterize clonal populations of T. pallidum subsp. pallidum Nichols, the most widely studied strain. In limiting dilutions experiments, it was possible to establish cultures with inocula as low as 0.5 T. pallidum per well despite the long generation time (~35 to 40 hours) of this organism. Six Nichols strain clones isolated by limiting dilution were characterized in detail. All clones exhibited indistinguishable morphology and motility, highly similar in vitro multiplication rates, and comparable infectivity in the rabbit model (ID50 ≤ 100 bacteria). Genomic sequencing revealed sequence heterogeneity in the form of insertions or deletions at 5 sites, single nucleotide variations at 20 sites, and polynucleotide (polyG/C) tract length differences at 22 locations. Genomic sequences of the uncloned Nichols strain preparations propagated in rabbits or in vitro cultures exhibited substantial heterogeneity at these locations, indicating coexistence of many varied 'clonotypes' within these populations. Nearly all genetic variations were specific for the Nichols strain and were not detected in the >280 T. pallidum genomic sequences that are currently available. We hypothesize that these Nichols strain-specific sequence variations arose independently either during human infection or within the 110 years since the strain's initial isolation, and thus represent examples of microevolution and divergence.

Treponema pallidum subsp. pallidum with an Artificially impaired TprK antigenic variation system is attenuated in the Rabbit model of syphilis.

BACKGROUND: The TprK protein of the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), undergoes antigenic variation in seven discrete variable (V) regions via non-reciprocal segmental gene conversion. These recombination events transfer information from a repertoire of 53 silent chromosomal donor cassettes (DCs) into the single tprK expression site to continually generate TprK variants. Several lines of research developed over the last two decades support the theory that this mechanism is central to T. pallidum's ability for immune avoidance and persistence in the host. Structural and modeling data, for example, identify TprK as an integral outer membrane porin with the V regions exposed on the pathogen's surface. Furthermore, infection-induced antibodies preferentially target the V regions rather than the predicted ß-barrel scaffolding, and sequence variation abrogates the binding of antibodies elicited by antigenically different V regions. Here, we engineered a T. pallidum strain to impair its ability to vary TprK and assessed its virulence in the rabbit model of syphilis. PRINCIPAL FINDINGS: A suicide vector was transformed into the wild-type (WT) SS14 T. pallidum isolate to eliminate 96% of its tprK DCs. The resulting SS14-DCKO strain exhibited an in vitro growth rate identical to the untransformed strain, supporting that the elimination of the DCs did not affect strain viability in absence of immune pressure. In rabbits injected intradermally with the SS14-DCKO strain, generation of new TprK sequences was impaired, and the animals developed attenuated lesions with a significantly reduced treponemal burden compared to control animals. During infection, clearance of V region variants originally in the inoculum mirrored the generation of antibodies to these variants, although no new variants were generated in the SS14-DCKO strain to overcome immune pressure. Naïve rabbits that received lymph node extracts from animals infected with the SS14-DCKO strain remained uninfected. CONCLUSION: These data further support the critical role of TprK in T. pallidum virulence and persistence during infection.

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.

[First case description of contagious ovine digital dermatitis in Switzerland]. / Erstbeschreibung der Krankheit «Kontagiöse ovine digitale Dermatitis¼ in der Schweiz.

INTRODUCTION: Contagious ovine digital dermatitis (CODD) is an emerging infectious foot disease in sheep. To date, CODD has been described in Great Britain, Ireland, Sweden and Germany and now in Switzerland for the first time. Unlike foot rot, the CODD lesions do not spread from the interdigital space, but usually begin at the dorsal/abaxial coronary band. The changes can spread to the hoof wall and the sole and finally can lead to exungulation, similar to foot rot. Treponema spp. are often found in CODD lesions analogous to digital dermatitis (Mortellaro's disease) in cattle. Involvement of Dichelobacter nodosus (D. nodosus) is considered a risk factor, but the presence of the bacterium is not mandatory. In February 2022, ulcerative lesions in the dorso-axial coronary band area were noticed on both claws of the left forelimb in an ewe. Histology of the biopsy showed hyperkeratosis and erosion with exocytosis and crust formation. Treponema spp. PCR and fluorescence in situ hybridization (FISH) were positive for Treponema phylotype 1 (PT1). In addition, D. nodosus and Porphyromonas levii could be detected in the biopsy using PCR. A single local application of chlortetracycline spray led to clinical healing within two weeks, no recurrence was seen within the following two months. Three control sheep, which were kept together with the diseased sheep, did not show any clinical signs of CODD. Treponema spp could not be found in interdigital and coronary band biopsies by PCR or FISH. This is the first description of CODD in Switzerland and aims to sensitize veterinarians to CODD as a differential diagnosis for foot diseases in sheep.

INTRODUCTION: La dermatite digitale contagieuse ovine (contagious ovine digital dermatitis; CODD) est une maladie infectieuse des onglons des moutons d'importance croissante. À ce jour, la CODD a été décrite en Grande-Bretagne, Irlande, Suède et Allemagne, et maintenant pour la première fois également en Suisse. Au contraire du piétain, les lésions de CODD ne s'étendent pas à partir de l'espace interdigité, mais elles commencent en général au bord coronaire dorsal/abaxial. De là, les lésions peuvent s'étendre à la corne de la paroi et à la sole, ce qui peut finalement conduire à une perte complète de la boite cornée de l'onglon, comme en cas de piétain. En analogie à la dermatite digitale (maladie de Mortellaro) chez les bovins, des tréponèmes sont souvent mis en évidence dans les lésions de CODD. La présence de Dichelobacter nodosus (D. nodosus) est considérée comme un facteur de risque, mais elle n'est pas indispensable au développement de la CODD. Des lésions ulcératives dans la région du bord coronaire dorso-axial des deux onglons antérieurs d'une brebis ont été remarqués en février 2022. L'examen histologique de la biopsie de la lésion de CODD a montré une hyperkératose ainsi que des érosions avec de l'exocytose et la formation de croûtes. Aussi bien la PCR pour les Treponema spp. que l'hybridisation in-situ à fluorescence (FISH) étaient positives pour Treponema Phylotype 1 (PT1). De plus, D. nodosus et Porphyromonas levii ont été mis en évidence dans la biopsie. Une application locale unique de spray à la tétracycline après le prélèvement de la biopsie a conduit à une guérison clinique en deux semaines, et aucune récidive n'a été observée dans le deux mois suivants. Trois moutons de boucherie qui étaient détenus avec la brebis malade mais ne présentaient pas de lésions de CODD ont servi de contrôles négatifs. Des Treponema spp. n'ont été mis en évidence chez ces animaux, ni dans des biopsies du bord coronaire ni dans celles de l'espace interdigité. Cette étude représente la première description de la CODD en Suisse et est destinée à sensibiliser la profession vétérinaire à la CODD comme diagnostic différentel en cas de maladies des onglons chez les moutons.

Associations between hair trace mineral concentrations and the occurrence of treponeme-associated hoof disease in elk (Cervus canadensis).

BACKGROUND: Trace minerals are important for animal health. Mineral deficiency or excess can negatively affect immune function, wound healing, and hoof health in domestic livestock, but normal concentrations and health impairment associated with mineral imbalances in wild animals are poorly understood. Treponeme-associated hoof disease (TAHD) is an emerging disease of free-ranging elk (Cervus canadensis) in the U.S. Pacific Northwest. Selenium and copper levels identified in a small number of elk from areas where TAHD is established (i.e., southwestern Washington) suggested a mineral deficiency may have increased susceptibility to TAHD. Our objectives were to determine trace mineral concentrations using hair from elk originating in TAHD affected areas of Washington, California, Idaho, and Oregon and assess their associations with the occurrence of the disease. RESULTS: We identified limited associations between TAHD occurrence and severity with hair mineral concentrations in 72 free-ranging elk, using Firth's logistic regression and multinomial regression models. We found consistent support for a priori hypotheses that selenium concentration, an important mineral for hoof health, is inversely associated with the occurrence of TAHD. Less consistent support was observed for effects of other minerals previously associated with hoof health (e.g., copper or zinc) or increased disease risk from potential toxicants. CONCLUSION: Trace mineral analysis of hair is a non-invasive sampling technique that offers feasibility in storage and collection from live animals and carcasses. For some minerals, levels in hair correlate with visceral organs that are challenging to obtain. Our study using hair collected opportunistically from elk feet submitted for diagnostic investigations provides a modest reference of hair mineral levels in elk from the U.S. Pacific Northwest that may be useful in future determination of reference ranges. Although our results revealed high variability in mineral concentrations between elk, consistent relationship of possibly low selenium levels and TAHD suggest that further investigations are warranted.

High frequency of Nichols-like strains and increased levels of macrolide resistance in Treponema pallidum in clinical samples from Buenos Aires, Argentina.

Globally, 94% of Treponema pallidum subsp. pallidum (TPA) clinical strains belong to the SS14-like group and 6% to the Nichols-like group, with a prevalence of macrolide resistance of 90%. Our goal was to determine whether local TPA strain distribution and macrolide resistance frequency have changed significantly since our last report, which revealed that Buenos Aires had a high frequency of Nichols-like strains (27%) and low levels of macrolide resistance (14%). Swab samples from patients with suspected syphilis were collected during 2015-2019 and loci TP0136, TP0548, TP0705 were sequenced in order to perform multilocus sequence typing. Strains were classified as Nichols-like or SS14-like. The presence of macrolide resistance-associated mutations was determined by examination of the 23S rDNA gene sequence. Of 46 typeable samples, 37% were classified as Nichols-like and 63% as SS14-like. Macrolide resistance prevalence was 45.7%. Seven allelic profiles were found, five were SS14-like and two were Nichols-like. The frequency of Nichols-like strains increased between studies (26.8% vs. 37%, p = 0.36). A dramatic increase was found in the frequency of macrolide resistant strains between studies (14.3% vs. 45.7%, p = 0.005). Our results are in agreement with international trends and underscore the need to pursue further TPA molecular typing studies in South America.

In Vitro Transformation and Selection of Treponema pallidum subsp. pallidum.

Although the isolation of Treponema pallidum subsp. pallidum (T. pallidum) from a syphilis patient dates to 1912, for the duration of the 20th century, this pathogen has remained an exceedingly difficult organism to study due to the lack of a system to support its viability in vitro. This limitation, in turn, has precluded the application of genetic engineering techniques via transformation and subsequent selection of T. pallidum transformants. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible for us to experiment with transformation and selection methods. Here we describe the approach that we adopted to successfully transform T. pallidum with foreign DNA and select the resulting recombinant strain using kanamycin. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Transformation of T. pallidum Support Protocol 1: Quantification of T. pallidum in suspensions using dark-field microscopy Support Protocol 2: Counting cells using a hemacytometer Basic Protocol 2: Selection, initial passaging, and expansion of transformed cultures Basic Protocol 3: Isolation of a clonal strain through limiting dilution.

A suite of PCR-LwCas13a assays for detection and genotyping of Treponema pallidum in clinical samples.

The performance of commonly used assays for diagnosis of syphilis varies considerably depending on stage of infection and sample type. In response to the need for improved syphilis diagnostics, we develop assays that pair PCR pre-amplification of the tpp47 gene of Treponema pallidum subsp. pallidum with CRISPR-LwCas13a. The PCR-LwCas13a assay achieves an order of magnitude better analytical sensitivity than real-time PCR with equivalent specificity. When applied to a panel of 216 biological specimens, including 135 clinically confirmed primary and secondary syphilis samples, the PCR-LwCas13a assay demonstrates 93.3% clinical sensitivity and 100% specificity, outperforming tpp47 real-time PCR and rabbit-infectivity testing. We further adapt this approach to distinguish Treponema pallidum subsp. pallidum lineages and identify genetic markers of macrolide resistance. Our study demonstrates the potential of CRISPR-based approaches to improve diagnosis and epidemiological surveillance of syphilis.