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.

Treponema peruense sp. nov., a commensal spirochaete isolated from human faeces.

A Gram-stain-negative, obligatory anaerobic spirochaete (RCC2812T) was isolated from a faecal sample obtained from an individual residing in a remote Amazonian community in Peru. The bacterium showed highest 16S rRNA gene sequence similarity to the pig intestinal spirochete Treponema succinifaciens (89.48 %). Average nucleotide identity values between strain RCC2812T and all available Treponema genomes from validated type strains were all <73 %, thus clearly lower than the species delineation threshold. The DNA G+C content of RCC2812T was 41.24 mol%. Phenotypic characterization using the API-ZYM and API 20A systems confirmed the divergent position of this bacterium within the genus Treponema. Strain RCC2812T could be differentiated from the phylogenetically most closely related T. succinifaciens by the presence of alkaline phosphatase and α -glucosidase activities. Unlike T. succinifaciens, strain RCC2812T grew equally well with or without serum. Strain RCC2812T is the first commensal Treponema isolated from the human faecal microbiota of remote populations, and based on the collected data represents a novel Treponema species for which the name Treponema peruense sp. nov. is proposed. The type strain is RCC2812T (=LMG 31794T=CIP 111910T).

Diversity of Treponema denticola and Other Oral Treponeme Lineages in Subjects with Periodontitis and Gingivitis.

More than 75 species/species-level phylotypes belonging to the genus Treponema inhabit the human oral cavity. Treponema denticola is commonly associated with periodontal disease, but the etiological roles and ecological distributions of other oral treponemes remain more obscure. Here, we compared the clinical distributions of phylogroup 1 and 2 oral treponemes in subgingival plaque sampled from Chinese subjects with periodontitis (n = 10) and gingivitis (n = 8) via sequence analysis of the highly conserved pyrH housekeeping gene. Two PCR primer sets that targeted oral phylogroup 1 and 2 treponeme pyrH genes were used to construct plasmid clone amplicon libraries for each subject, and the libraries were sequenced for bioinformatic analysis. A total of 1,204 quality-filtered, full-length pyrH gene sequences were obtained from the cohort (median number, 61.5 cloned pyrH sequences per subject; range, 59 to 83), which were assigned to 34 pyrH genotypes (designated pyrH001 to pyrH034; 97% sequence identity cutoff). Eighteen pyrH genotypes (536 pyrH sequences) corresponded to phylogroup 1 treponeme taxa (including Treponema vincentii and Treponema medium). Sixteen pyrH genotypes (668 pyrH sequences) corresponded to T. denticola and other phylogroup 2 treponemes. Samples from periodontitis subjects contained a greater diversity of phylogroup 2 pyrH genotypes than did samples from gingivitis subjects (Mann-Whitney U test). One T. denticola pyrH genotype (pyrH001) was highly prevalent, detected in 10/10 periodontitis and 6/8 gingivitis subjects. Several subjects harbored multiple T. denticola pyrH genotypes. Nonmetric multidimensional scaling and permutational multivariate analysis of variance (PERMANOVA) revealed no significant differences in overall pyrH genotype compositions between periodontitis and gingivitis subjects. Taken together, our results show that subjects with periodontitis and gingivitis commonly harbor highly taxonomically diverse communities of oral treponemes. IMPORTANCE Periodontal diseases, such as periodontitis, are highly complex, multifactorial inflammatory infectious diseases affecting the gums and tooth-supporting structures. They are caused by chronic accumulations of dental plaque below the gum line that typically comprise hundreds of different bacterial species. Certain species of spiral-shaped bacteria known as treponemes, most notably Treponema denticola, are proposed to play key roles in the development and progression of periodontal disease. In our study, we characterized the genetic lineages of T. denticola, Treponema vincentii, Treponema medium, and related species of treponeme bacteria that were present in dental plaque samples from Chinese subjects with periodontal disease. Our results revealed that individual subjects commonly harbored multiple genetic lineages (strains) of T. denticola and other species of treponeme bacteria. Taken together, our results indicate that highly diverse and complex populations of oral treponemes may be present in dental plaque, which may potentially play important roles affecting periodontal health status.

Multilocus sequence typing of Treponema pallidum subsp. pallidum in Barcelona.

Aim: To implement the multilocus sequence typing (MLST) methodology in syphilis samples previously characterized by enhanced CDC typing (ECDCT) and macrolide resistance. Materials & methods: MLST was performed on genital ulcer and blood samples by analyzing a region of the tp0136, tp0548 and tp0705loci using Sanger sequencing. Results: Up to 59/85 (69.4%) of genital ulcer and 4/39 (10.3%) of whole blood samples were fully typed. The most frequent profiles were 1.3.1 (56%) and 1.1.1 (11%). All the 1.3.1 samples typed carried the A2058G mutation, responsible for macrolide resistance. MLST and ECDCT showed similar overall typing yields. Conclusion: Several allelic profiles of T. pallidum subsp. pallidum were identified and classified into two major genetic clades in Barcelona. Our results were similar to that described in Europe.

First cases of contagious ovine digital dermatitis in Germany.

Contagious ovine digital dermatitis (CODD) is a significant disease of the ovine foot characterised by severe lameness and progressive separation of the hoof horn capsule from the underlying tissue. Similar to bovine digital dermatitis (BDD), pathogenic members of the genus Treponema including the Treponema medium phylogroup, Treponema phagedenis phylogroup and Treponema pedis are frequently found together in CODD lesions. To date, CODD was only described in Ireland and the United Kingdom. In northern Germany, cases of an unusually severe lameness presented in a sheep flock that had been affected by footrot for several years. These cases were non-responsive to conventional footrot therapies, with some sheep exhibiting substantial lesions of the claw horn that resulted in horn detachment. Lesion swab samples were collected from both clinically affected and asymptomatic animals. In all clinically affected sheep, CODD-associated Treponema phylogroups were detected by polymerase chain reaction. This is the first report of CODD in Germany and mainland Europe, indicating a wider geographic spread than previously considered. In cases of severe lameness attributed to claw lesions in sheep that fail to respond to footrot treatment, CODD should be considered irrespective of geographic location.

Treponema spp., the dominant pathogen in the lesion of bovine digital dermatitis and its characterization in dairy cattle.

This study examined the presence of Treponema in lesions using conventional PCR detection methods and investigated the microbiome by performing high-throughput DNA sequencing. Twenty-nine bovine digital dermatitis (BDD) lesions were collected from 25 dairy farms in South Korea that were tested by PCR amplification using sets of one universal, one genus-specific, and three species specific Treponema PCR primers. Three BDD samples were randomly selected and normal tissue samples were submitted for 16S rRNA sequencing using the Illumina MiSeq platform. The dominant phylum present in all tested BDD lesions was Spirochaetes with a mean relative abundance of 46.9 %, and Treponema was the most abundant genus. Spirochaetes abundance was followed by the phyla Tenericutes and Bacteroidetes with 14.1 % and 11.8 % mean abundances, respectively. Co-infecting bacteria from phyla Tenericutes and Bacteroidetes may be involved in the progression of BDD. Bovine digital dermatitis infection is polymicrobial in nature, but Treponema spp. are the main etiologic agents of the disease. In the microbiome results, Treponema pedis had the highest mean relative abundance (20.9 %) in the BDD lesions in this study followed by T. denticola, T. medium, T. lecithinolyricum, Spirochaeta africana, and Sediminispirochaeta bajacalifoniensis. All 29 samples were positive in the genus-specific Treponema PCR results. The species-specific PCR resulted in 75.9 %, 86.2 %, and 69.0 % of samples in groups T. medium/T. vincentii-like, T. phagedenis-like, and T. pedis, respectively. Understanding how these microorganisms mutually interact in the host during certain stages of infection may help in the development of better practices for controlling BDD.

Comparative genomics and full-length Tprk profiling of Treponema pallidum subsp. pallidum reinfection.

Developing a vaccine against Treponema pallidum subspecies pallidum, the causative agent of syphilis, remains a public health priority. Syphilis vaccine design efforts have been complicated by lack of an in vitro T. pallidum culture system, prolific antigenic variation in outer membrane protein TprK, and lack of functional annotation for nearly half of the genes. Understanding the genetic basis of T. pallidum reinfection can provide insights into variation among strains that escape cross-protective immunity. Here, we present comparative genomic sequencing and deep, full-length tprK profiling of two T. pallidum isolates from blood from the same patient that were collected six years apart. Notably, this patient was diagnosed with syphilis four times, with two of these episodes meeting the definition of neurosyphilis, during this interval. Outside of the highly variable tprK gene, we identified 14 coding changes in 13 genes. Nine of these genes putatively localized to the periplasmic or outer membrane spaces, consistent with a potential role in serological immunoevasion. Using a newly developed full-length tprK deep sequencing protocol, we profiled the diversity of this gene that far outpaces the rest of the genome. Intriguingly, we found that the reinfecting isolate demonstrated less diversity across each tprK variable region compared to the isolate from the first infection. Notably, the two isolates did not share any full-length TprK sequences. Our results are consistent with an immunodominant-evasion model in which the diversity of TprK explains the ability of T. pallidum to successfully reinfect individuals, even when they have been infected with the organism multiple times.

Treponema phagedenis (ex Noguchi 1912) Brumpt 1922 sp. nov., nom. rev., isolated from bovine digital dermatitis.

'Treponema phagedenis' was originally described in 1912 by Noguchi but the name was not validly published and no type strain was designated. The taxon was not included in the Approved Lists of Bacterial Names and hence has no standing in nomenclature. Six Treponema strains positive in a 'T. phagedenis' phylogroup-specific PCR test were isolated from digital dermatitis (DD) lesions of cattle and further characterized and compared with the human strain 'T. phagedenis' ATCC 27087. Results of phenotypic and genotypic analyses including API ZYM, VITEK2, MALDI-TOF and electron microscopy, as well as whole genome sequence data, respectively, showed that they form a cluster of species identity. Moreover, this species identity was shared with 'T. phagedenis'-like strains reported in the literature to be regularly isolated from bovine DD. High average nucleotide identity values between the genomes of bovine and human 'T. phagedenis' were observed. Slight genomic as well as phenotypic variations allowed us to differentiate bovine from human isolates, indicating host adaptation. Based on the fact that this species is regularly isolated from bovine DD and that the name is well dispersed in the literature, we propose the species Treponema phagedenis sp. nov., nom. rev. The species can phenotypically and genetically be identified and is clearly separated from other Treponema species. The valid species designation will allow to further explore its role in bovine DD. The type strain for Treponema phagedenis sp. nov., nom. rev. is B43.1T (=DSM 110455T=NCTC 14362T) isolated from a bovine DD lesion in Switzerland.

Strain diversity of Treponema pallidum subsp. pertenue suggests rare interspecies transmission in African nonhuman primates.

In our most recent study, we found that in Tanzania infection with Treponema pallidum (TP) subsp. pertenue (TPE) is present in four different monkey species. In order to gain information on the diversity and epidemiological spread of the infection in Tanzanian nonhuman primates (NHP), we identified two suitable candidate genes for multi-locus sequence typing (MLST). We demonstrate the functionality of the MLST system in invasively and non-invasively collected samples. While we were not able to demonstrate frequent interspecies transmission of TPE in Tanzanian monkeys, our results show a clustering of TPE strains according to geography and not host species, which is suggestive for rare transmission events between different NHP species. In addition to the geographic stability, we describe the relative temporal stability of the strains infecting NHPs and identified multi-strain infection. Differences between TPE strains of NHP and human origin are highlighted. Our results show that antibiotic resistance does not occur in Tanzanian TPE strains of NHP origin.

Passive Filtration, Rapid Scanning Electron Microscopy, and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Treponema Culture and Identification from the Oral Cavity.

We present here a new passive-filtration-based culture device combined with rapid identification with a new electron microscope (Hitachi TM4000) for the detection and culture of Treponema species from the human oral cavity. Of the 44 oral samples cultivated, 15 (34%) were found to be positive for Treponema using electron microscopy and were also culture positive. All were subcultured on agar plates; based on genome sequencing and analyses, 10 were strains of Treponema pectinovorum and 5 were strains of Treponema denticola The 29 samples that were negative for Treponema remained culture negative. In addition, 14 Treponema species ordered from the DSMZ collection were cultured in the T-Raoult culture medium optimized here. Finally, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used and 30 novel spectra were added to the MALDI-TOF MS database. We have successfully developed a new and effective method for treponemal detection, culture, and identification.

Comparative genomics of the transportome of Ten Treponema species.

Treponema is a diverse bacterial genus, the species of which can be pathogenic, symbiotic, or free living. These treponemes can cause various diseases in humans and other animals, such as periodontal disease, bovine digital dermatitis and animal skin lesions. However, the most important and well-studied disease of treponemes that affects humans is 'syphilis'. This disease is caused by Treponema pallidum subspecie pallidum with 11-12 million new cases around the globe on an annual basis. In this study we analyze the transportome of ten Treponema species, with emphasis on the types of encoded transport proteins and their substrates. Of the ten species examined, two (T. primitia and T. azonutricium) reside as symbionts in the guts of termites; six (T. pallidum, T. paraluiscuniculi, T. pedis, T. denticola, T. putidum and T. brennaborense) are pathogens of either humans or animals, and T. caldarium and T. succinifaciens are avirulent species, the former being thermophilic. All ten species have a repertoire of transport proteins that assists them in residing in their respective ecological niches. For instance, oral pathogens use transport proteins that take up nutrients uniquely present in their ecosystem; they also encode multiple multidrug/macromolecule exporters that protect against antimicrobials and aid in biofilm formation. Proteins of termite gut symbionts convert cellulose into other sugars that can be metabolized by the host. As often observed for pathogens and symbionts, several of these treponemes have reduced genome sizes, and their small genomes correlate with their dependencies on the host. Overall, the transportomes of T. pallidum and other pathogens have a conglomerate of parasitic lifestyle-assisting proteins. For example, a T. pallidum repeat protein (TprK) mediates immune evasion; outer membrane proteins (OMPs) allow nutrient uptake and end product export, and several ABC transporters catalyze sugar uptake, considered pivotal to parasitic lifestyles. Taken together, the results of this study yield new information that may help open new avenues of treponeme research.

Associations between digital dermatitis lesion grades in dairy cattle and the quantities of four Treponema species.

Digital dermatitis (DD) presents as painful, ulcerative or proliferative lesions that lead to bovine lameness affecting economic efficiency and animal welfare. Although DD etiological agent(s) have not been established, it is widely accepted that DD is a polymicrobial disease significantly associated with species of Treponema and the non-linear disease progression may be attributed to interactions among infecting bacteria. We postulated the morphological changes associated with DD lesion grades are related to interactions among infecting species of Treponema. We developed a novel species-specific qPCR that can identify the absolute abundance of the four of the most common species of Treponema in DD, T. phagedenis, T. medium, T. pedis and T. denticola, in a single reaction. We found species abundance and the number of distinct Treponema species present is higher in active, ulcerative lesions than in healing lesions, chronic lesions, and DD-free skin. Treponema spp. were present in both DD-free skin and M3 lesions following treatment with oxytetracycline. We have also found positive correlations among T. phagedenis, T. medium and T. pedis indicating they are significantly more likely to be found together than apart and their absolute quantities tend to increase together, a relationship which is not present with T. denticola. Further, we found Treponema, particularly viable T. denticola, in lesions 5 days post treatment with oxytetracycline (M3). Our findings suggest that pathogenicity may be closely associated with Treponema abundance, particularly T. phagedenis, T. medium and T. pedis, and interactions among them, independent of T. denticola. Our results provide a novel, consistent method to identify species of Treponema within DD lesions and associate Treponema spp. and abundance with morphological changes related to host pathogenicity.

The bacterial and archaeal community structures and methanogenic potential of the cecal microbiota of goats fed with hay and high-grain diets.

The cecum plays an important role in the feed fermentation of ruminants. However, information is very limited regarding the cecal microbiota and their methane production. In the present study, the cecal content from twelve local Chinese goats, fed with either a hay diet (0% grain) or a high-grain diet (71.5% grain), were used to investigate the bacterial and archaeal community and their methanogenic potential. Microbial community analysis was determined using high-throughput sequencing of 16S rRNA genes and real-time PCR, and the methanogenesis potential was assessed by in vitro fermentation with ground corn or hay as substrates. Compared with the hay group, the high-grain diet significantly increased the length and weight of the cecum, the proportions of starch and crude protein, the concentrations of volatile fatty acids and ammonia nitrogen, but decreased the pH values (P < 0.05). The high-grain diet significantly increased the abundances of bacteria and archaea (P < 0.05) and altered their community. For the bacterial community, the genera Bifidobacterium, Prevotella, and Treponema were significantly increased in the high-grain group (P < 0.05), while Akkermansia, Oscillospira, and Coprococcus were significantly decreased (P < 0.05). For the archaeal community, Methanosphaera stadtmanae was significantly increased in the high-grain group (P < 0.05), while Methanosphaera sp. ISO3-F5 was significantly decreased (P < 0.05). In the in vitro fermentation with grain as substrate, the cecal microorganisms from the high-grain group produced a significantly higher amount of methane and volatile fatty acids (P < 0.05), and produced significantly lower amount of lactate (P < 0.05). Conclusively, high-grain diet led to more fermentable substrates flowing into the hindgut of goats, resulting in an enhancement of microbial fermentation and methane production in the cecum.

Antimicrobial susceptibility profiles of oral Treponema species.

Treponemes occur in the microflora of the dental plaque. Certain Treponema species that are frequently isolated from chronic periodontitis lesions are involved in its initiation and progression. In addition to mechanical instrumentation, antimicrobial agents are used as an adjunctive treatment modality for periodontitis. Despite its importance for successful antimicrobial treatment, information about susceptibility is limited for Treponema species. The aim of this study was to assess the susceptibility of Treponema denticola strains, Treponema socranskii, and Treponema vincentii to eleven antimicrobial agents. The minimum inhibitory and minimum bactericidal concentrations of these antimicrobial agents revealed strain-specific variation. Doxycycline, minocycline, azithromycin, and erythromycin were effective against all Treponema species tested in this study, whereas fluoroquinolones only exhibited an equivalent effectiveness on T. socranskii. The susceptibility of one T. denticola strain, T. socranskii, and T. vincentii to kanamycin was influenced by prior exposure to aerobic conditions. The susceptibility to quinolone drugs varied among strains of T. denticola, although they share an amino acid sequence identity of greater than 99% for DNA gyrase (type II topoisomerase) subunit A. In addition, an ATP-binding cassette (ABC) transporter inhibitor assay for T. denticola indicated that the transport of quinolone drugs is partially related to this transporter, although there may be parallel transport mechanisms. Our results provide important insights into antimicrobial agent-Treponema dynamics and establish a basis for developing an appropriate adjunctive therapy for periodontal disease.

Treponema rectale sp. nov., a spirochete isolated from the bovine rectum.

A Gram-stain-negative, obligatory anaerobic spirochete, CHPAT, was isolated from the rectal tissue of a Holstein-Friesian cow. On the basis of 16S rRNA gene comparisons, CHPAT was most closely related to the human oral spirochete, Treponema parvum, with 88.8 % sequence identity. Further characterisation on the basis of recA gene sequence analysis, cell morphology, pattern of growth and physiological profiling identified marked differences with respect to other recognised species of the genus Treponema. Microscopically, the helical cells measured approximately 1-5 µm long and 0.15-0.25 µm wide, with two to five irregular spirals. Transmission electron microscopy identified four periplasmic flagella in a 2 : 4 : 2 arrangement. CHPAT grew independently of serum, demonstrated no evidence of haemolytic activity and possessed an in vitro enzyme activity profile that is unique amongst validly named species of the genus Treponema, exhibiting C4 esterase, α-galactosidase and ß-galactosidase activity. Taken together, these data indicate that CHPAT represents a novel species of the genus Treponema, for which the name Treponema rectale is proposed. The type strain of Treponema rectale is CHPAT (=DSM 103679T=NCTC 13848T).

Oral treponeme major surface protein: Sequence diversity and distributions within periodontal niches.

Treponema denticola and other species (phylotypes) of oral spirochetes are widely considered to play important etiological roles in periodontitis and other oral infections. The major surface protein (Msp) of T. denticola is directly implicated in several pathological mechanisms. Here, we have analyzed msp sequence diversity across 68 strains of oral phylogroup 1 and 2 treponemes; including reference strains of T. denticola, Treponema putidum, Treponema medium, 'Treponema vincentii', and 'Treponema sinensis'. All encoded Msp proteins contained highly conserved, taxon-specific signal peptides, and shared a predicted 'three-domain' structure. A clone-based strategy employing 'msp-specific' polymerase chain reaction primers was used to analyze msp gene sequence diversity present in subgingival plaque samples collected from a group of individuals with chronic periodontitis (n=10), vs periodontitis-free controls (n=10). We obtained 626 clinical msp gene sequences, which were assigned to 21 distinct 'clinical msp genotypes' (95% sequence identity cut-off). The most frequently detected clinical msp genotype corresponded to T. denticola ATCC 35405T , but this was not correlated to disease status. UniFrac and libshuff analysis revealed that individuals with periodontitis and periodontitis-free controls harbored significantly different communities of treponeme clinical msp genotypes (P<.001). Patients with periodontitis had higher levels of clinical msp genotype diversity than periodontitis-free controls (Mann-Whitney U-test, P<.05). The relative proportions of 'T. vincentii' clinical msp genotypes were significantly higher in the control group than in the periodontitis group (P=.018). In conclusion, our data clearly show that both healthy and diseased individuals commonly harbor a wide diversity of Treponema clinical msp genotypes within their subgingival niches.

Treponema ruminis sp. nov., a spirochaete isolated from the bovine rumen.

A novel bacterium, strain Ru1T, was encountered during a survey of spirochaetes living in the gastrointestinal tract of ruminants. Comparative analysis of 16S rRNA gene sequence data indicated that strain Ru1T clustered within the genus Treponemabut shared at most 86.1 % sequence similarity with other recognised species of the genus Treponema. Further phylogenetic analysis based on partial recombinase A (recA) gene sequence comparisons, together with phenotypic characterization, also demonstrated the divergence of strain Ru1T from other recognised species of the genus Treponema. Microscopically, strain Ru1T appeared as a very small, highly motile, helical spirochaete with eight periplasmic flagella in a 4 : 8 : 4 arrangement. It exhibited C8 esterase lipase, leucine arylamidase, ß-galactosidase and ß-glucosidase activity. A distinctive, serum-independent growth pattern was also observed, characterized by colonies with an absence of the local haemolysis that is typical of many pathogenic treponemes. On the basis of these data, strain Ru1T is considered to represent a novel species of the genus Treponema, for which the name Treponema ruminis sp. nov. is proposed. The type strain is Ru1T (=DSM 103462T=NCTC 13847T).

Multilocus Sequence Analysis of Phylogroup 1 and 2 Oral Treponeme Strains.

More than 75 "species-level" phylotypes of spirochete bacteria belonging to the genus Treponema reside within the human oral cavity. The majority of these oral treponeme phylotypes correspond to as-yet-uncultivated taxa or strains of uncertain standing in taxonomy. Here, we analyze phylogenetic and taxonomic relationships between oral treponeme strains using a multilocus sequence analysis (MLSA) scheme based on the highly conserved 16S rRNA, pyrH, recA, and flaA genes. We utilized this MLSA scheme to analyze genetic data from a curated collection of oral treponeme strains (n = 71) of diverse geographical origins. This comprises phylogroup 1 (n = 23) and phylogroup 2 (n = 48) treponeme strains, including all relevant American Type Culture Collection reference strains. The taxonomy of all strains was confirmed or inferred via the analysis of ca. 1,450-bp 16S rRNA gene sequences using a combination of bioinformatic and phylogenetic approaches. Taxonomic and phylogenetic relationships between the respective treponeme strains were further investigated by analyzing individual and concatenated flaA (1,074-nucleotide [nt]), recA (1,377-nt), and pyrH (696-nt) gene sequence data sets. Our data confirmed the species differentiation between Treponema denticola (n = 41) and Treponema putidum (n = 7) strains. Notably, our results clearly supported the differentiation of the 23 phylogroup 1 treponeme strains into five distinct "species-level" phylotypes. These respectively corresponded to "Treponema vincentii" (n = 11), Treponema medium (n = 1), "Treponema sinensis" (Treponema sp. IA; n = 4), Treponema sp. IB (n = 3), and Treponema sp. IC (n = 4). In conclusion, our MLSA-based approach can be used to effectively discriminate oral treponeme taxa, confirm taxonomic assignment, and enable the delineation of species boundaries with high confidence. IMPORTANCE: Periodontal diseases are caused by persistent polymicrobial biofilm infections of the gums and underlying tooth-supporting structures and have a complex and variable etiology. Although Treponema denticola is strongly associated with periodontal diseases, the etiological roles of other treponeme species/phylotypes are less well defined. This is due to a paucity of formal species descriptions and a poor understanding of genetic relationships between oral treponeme taxa. Our study directly addresses these issues. It represents one of the most comprehensive analyses of oral treponeme strains performed to date, including isolates from North America, Europe, and Asia. We envisage that our results will greatly facilitate future metagenomic efforts aimed at characterizing the clinical distributions of oral treponeme species/phylotypes, helping investigators to establish a more detailed understanding of their etiological roles in periodontal diseases and other infectious diseases. Our results are also directly relevant to various polymicrobial tissue infections in animals, which also involve treponeme populations.

Multilocus Sequence Typing of Pathogenic Treponemes Isolated from Cloven-Hoofed Animals and Comparison to Treponemes Isolated from Humans.

UNLABELLED: Treponema species are implicated in many diseases of humans and animals. Digital dermatitis (DD) treponemes are reported to cause severe lesions in cattle, sheep, pigs, goats, and wild elk, causing substantial global animal welfare issues and economic losses. The fastidiousness of these spirochetes has previously precluded studies investigating within-phylogroup genetic diversity. An archive of treponemes that we isolated enabled multilocus sequence typing to quantify the diversity and population structure of DD treponemes. Isolates (n = 121) were obtained from different animal hosts in nine countries on three continents. The analyses herein of currently isolated DD treponemes at seven housekeeping gene loci confirm the classification of the three previously designated phylogroups: the Treponema medium, Treponema phagedenis, and Treponema pedis phylogroups. Sequence analysis of seven DD treponeme housekeeping genes revealed a generally low level of diversity among the strains within each phylogroup, removing the need for the previously used "-like" suffix. Surprisingly, all isolates within each phylogroup clustered together, regardless of host or geographic origin, suggesting that the same sequence types (STs) can infect different animals. Some STs were derived from multiple animals from the same farm, highlighting probable within-farm transmissions. Several STs infected multiple hosts from similar geographic regions, identifying probable frequent between-host transmissions. Interestingly, T. pedis appears to be evolving more quickly than the T. medium or T. phagedenis DD treponeme phylogroup, by forming two unique ST complexes. The lack of phylogenetic discrimination between treponemes isolated from different hosts or geographic regions substantially contrasts with the data for other clinically relevant spirochetes. IMPORTANCE: The recent expansion of the host range of digital dermatitis (DD) treponemes from cattle to sheep, goats, pigs, and wild elk, coupled with the high level of 16S rRNA gene sequence similarity across hosts and with human treponemes, suggests that the same bacterial species can cause disease in multiple different hosts. This multilocus sequence typing (MLST) study further demonstrates that these bacteria isolated from different hosts are indeed very similar, raising the potential for cross-species transmission. The study also shows that infection spread occurs frequently, both locally and globally, suggesting transmission by routes other than animal-animal transmission alone. These results indicate that on-farm biosecurity is important for controlling disease spread in domesticated species. Continued surveillance and vigilance are important for ascertaining the evolution and tracking any further host range expansion of these important pathogens.

Isolation of digital dermatitis treponemes from cattle hock skin lesions.

BACKGROUND: Bovine hock lesions present a serious welfare and production issue on dairy farms worldwide. Current theories suggest that trauma is an important factor in the formation of hock lesions, although infection may also play a role in increasing their severity and duration. HYPOTHESIS: Digital dermatitis (DD) lesions in dairy cows are strongly associated with specific treponeme bacteria which are opportunistic invaders of other skin regions. Hock lesions were tested to ascertain if they too contained treponemes. ANIMALS: Swab and tissue samples were taken from hock lesions from two farms in South West England. METHODS: Hock lesions were classified into two categories: open lesions, which were often bleeding and ulcerated, or were encrusted; and closed lesions, which were classified as hair loss with no skin breakage. PCR assays and bacterial isolation were used to detect treponemes in hock lesions. RESULTS: All three phylogroups of digital dermatitis treponemes were detectable and isolated from open hock lesions only, with closed lesions showing no evidence of treponeme infection, either by PCR or bacterial culture. When analysed by 16S rRNA gene sequencing, the cultured treponeme DNA showed complete homology or was very similar to that found in foot lesions. Additionally, skin swabs from near the open hock wounds were also positive by PCR assay and isolation for the DD treponemes. CONCLUSIONS AND CLINICAL IMPORTANCE: Identification of the contribution of these infectious agents will allow for more optimal treatments to be developed that reduce the prevalence and healing times of both hock and DD lesions.