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.

Genome-wide association and functional genomic analyses for various hoof health traits in North American Holstein cattle.

Hoof diseases are a major welfare and economic issue in the global dairy cattle production industry, which can be minimized through improved management and breeding practices. Optimal genetic improvement of hoof health could benefit from a deep understanding of the genetic background and biological underpinning of indicators of hoof health. Therefore, the primary objectives of this study were to perform genome-wide association studies, using imputed high-density genetic markers data from North American Holstein cattle, for 8 hoof-related traits: digital dermatitis, sole ulcer, sole hemorrhage, white line lesion, heel horn erosion, interdigital dermatitis, interdigital hyperplasia, and toe ulcer, and a hoof health index. De-regressed estimated breeding values from 25,580 Holstein animals were used as pseudo-phenotypes for the association analyses. The genomic quality control, genotype phasing, and genotype imputation were performed using the PLINK (version 1.9), Eagle (version 2.4.1), and Minimac4 software, respectively. The functional genomic analyses were performed using the GALLO R package and the DAVID platform. We identified 22, 34, 14, 22, 28, 33, 24, 43, and 15 significant markers for digital dermatitis, heel horn erosion, interdigital dermatitis, interdigital hyperplasia, sole hemorrhage, sole ulcer, toe ulcer, white line lesion disease, and the hoof health index, respectively. The significant markers were located across all autosomes, except BTA10, BTA12, BTA20, BTA26, BTA27, and BTA28. Moreover, the genomic regions identified overlap with various previously reported quantitative trait loci for exterior, health, meat and carcass, milk, production, and reproduction traits. The enrichment analyses identified 44 significant gene ontology terms. These enriched genomic regions harbor various candidate genes previously associated with bone development, metabolism, and infectious and immunological diseases. These findings indicate that hoof health traits are highly polygenic and influenced by a wide range of biological processes.

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.

Prevalence of digital dermatitis using mirror scoring in Finnish freestall dairy herds.

Digital dermatitis (DD) is a severe bacterial hoof disease found worldwide. The disease can be classified into 5 different stages, denoted as M1 to M4 and M4.1, by clinical examination. The main objective of this study was to estimate prevalence of DD lesions in Finnish freestall dairy cattle population through hind feet inspection of standing cows with a mirror. Another aim was to estimate the sensitivity and specificity of mirror scoring on standing cows in a pen or in a milking parlor without washing the feet. Three veterinarians visited 81 randomly selected herds across the country. During the herd visits, hind feet of standing cows (n = 7,010) were scored with a mirror without washing the feet, either when the cows were standing in a pen (n = 4,992) or in the milking parlor (n = 2018). In total, 128 cows (111 from pen and 17 from milking parlor) including 256 feet were chosen with cross-sectional sampling and scored in a trimming chute. Animal-level sensitivity for scoring M2 lesions with a mirror was 55% and specificity was 97%; for all active DD lesions (M1, M2, or M4.1), sensitivity was 36% and specificity was 96%. Sensitivity for scoring any DD lesions was 90% and specificity was 82%. The bias-corrected sensitivity and specificity for scoring any DD lesions were 79% and 92%, respectively. The bias-corrected sensitivity and specificity for scoring M2 DD lesions were 10% and 100%. We found M2 lesions in 12.1% of the study herds, and true herd-level prevalence was the same. Altogether, 33.3% (true prevalence 28.4%) of the herds had either M1, M2, or M4.1 DD lesions. However, only 0.7% (true prevalence 5.4%) of cows in total had active M2 lesions. The within-herd prevalence of M2 lesions (in herds where at least 1 cow had a M2 lesion) was 5.7% and varied between 0.4% and 18.8%. Herds with active DD lesions also had more any DD lesions than herds without active DD lesions. The herd-level prevalence was higher than previously thought, with only 1 herd without any DD lesions. However, the animal-level prevalence of active DD lesions was relatively low. Farmers and veterinarians need to be informed of the disease and possible control measures. Because of the low within-herd prevalence, the control of the disease might be easier than in countries where DD is widespread. Further studies are needed to identify factors associated with DD prevalence in Finnish dairy herds.

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.

Short communication: Anesthetic residues in milk after topical application during treatment of hoof lesions in dairy cows.

Hoof lesions in dairy cows are usually treated by trimming the hoof. However, trimming by itself can cause severe pain or exacerbate already existing pain. Hoof trimming is usually not carried out by trained veterinarians, and pain management is not provided. Pain control during trimming is not only an ethical obligation but also allows for better manipulation and more meticulous treatment. Tri-Solfen (Bayer Animal Health, Pymble, Australia) is a spray gel containing lidocaine, bupivacaine, and cetrimide that is easily applied topically and has demonstrated pain-mitigation effects during and after hoof trimming. In the European Union, these local anesthetics are not approved for use in food-producing animals because of a lack of residue data and concerns about genotoxic effects in cattle and humans. The aim of this study was to assess lidocaine, bupivacaine, and 2,6-xylidine residues in milk after Tri-Solfen application in dairy cows. Five dairy cattle in the dry-off period were enrolled in the study based on clinical evidence of lameness (score ≥3 on a 5-point scale). After cleaning and superficial trimming, we applied 3 to 14 mL of Tri-Solfen to the lesions before continuing treatment. Two milk samples were collected per animal in the following 4 milkings and analyzed in a reference laboratory. Residues of lidocaine above the limits of quantification (0.2 µg/L) were found in milk samples in the first milking 6 h after treatment in only 2 cows. This study shows that excretion of local anesthetics and their metabolites in milk after topical application of Tri-Solfen is negligible and even undetectable after the first milking 6 h post-treatment.

Treponeme-Associated Hoof Disease of Free-Ranging Elk ( Cervus elaphus) in Southwestern Washington State, USA.

A novel foot disease in free-ranging elk ( Cervus elaphus) in southwestern Washington State emerged in 2008 and spread throughout the region. Initial studies showed adult elk had chronic hoof overgrowth, sole ulcers, and sloughed hoof capsules, but no cause was determined. To identify possible causes and characterize the earliest lesions, 9-, 7-, and 3-month-old elk were collected. Nine-month-old elk had sole ulcers (3/9 elk) and sloughed/overgrown hoof capsules (4/9 elk) similar to adults. Histologically, lesions consisted of coronary, heel bulb, and interdigital ulcers with suppurative inflammation, epithelial hyperplasia, deeply invasive spirochetes, and underrunning of the hoof capsule and heel-sole junction. Spirochetes were identified as Treponema via immunohistochemistry and polymerase chain reaction (PCR). Seven-month-old elk had similar underrunning foot ulcers (6/8 elk) with Treponema identified in all lesions but no chronic overgrowth or sloughed hoof capsules. Three-month-old calves had superficial coronary erosions with no inflammation or identifiable spirochetes (3/5 elk) but were culture/PCR positive for Treponema, suggesting possible early lesions. Lesions from 9- and 7-month-old elk included aerobic and anaerobic bacteria, many of which are associated with infectious foot disease in livestock. Antibody enzyme-linked immunosorbent assay of 7- and 3-month-old elk from the enzootic region showed a trend toward increased Treponema antibody titers compared to normal control elk from outside the region, further supporting the significance of Treponema in the pathogenesis of foot disease. Treponeme-associated hoof disease (TAHD) in elk, a debilitating and progressive condition, shares similarities to bovine digital dermatitis and contagious ovine digital dermatitis.

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.

Correlation of lesion severity with bacterial changes in Treponeme-Associated Hoof Disease from free-roaming wild elk (Cervus canadensis).

BACKGROUND: Treponeme-Associated Hoof Disease (TAHD) is a polybacterial, multifactorial disease affecting free-ranging wild elk (Cervus canadensis) in the Pacific Northwest. Previous studies have indicated a bacterial etiology similar to digital dermatitis in livestock, including isolation of Treponema species from lesions. The lesions appear to progress rapidly from ulcerative areas in the interdigital space or along the coronary band to severe, ulcerative, necrotic, proliferative lesions under-running the hoof wall, perforating the sole, and contributing to hoof elongation, deformity, and overgrowth. Eventually the lesions undermine the laminal structure leading to sloughing of the hoof horn capsule. The objective of this study was to characterize the bacterial communities associated with hoof lesions, which were categorized into 5 stages or disease grade severities, with 0 being unaffected tissue and 4 being sloughed hoof capsule. We also wanted to determine if the etiology of TAHD through morphological changes was dominated by Treponema, as observed in hoof diseases in livestock. RESULTS: The bacterial 16S rRNA gene was sequenced from 66 hoof skin biopsy samples representing 5 lesion grades from samples collected by Washington Department of Fish and Wildlife as part of a voluntary hunter program. Analysis of the relative abundance of bacterial sequences showed that lesions were dominated by members of the bacterial phyla Proteobacteria, Firmicutes, Spirochaetes, Bacteroidetes and Actinobacteria. In lesion samples, members of the genus Treponema, Porphyromonas, and Mycoplasma increased with lesion severity. Association analysis indicated frequent identification of Treponema with Porphyromonas, Bacteroides and other anaerobic Gram-positive cocci. CONCLUSIONS: The bacterial 16S rRNA gene sequencing confirmed the presence of Treponema species at all stages of TAHD lesions, treponeme specie-specific PCR and histopathology, indicating that the morphological changes are a continual progression of disease severity with similar bacterial communities. Association and abundance of these other pathogenic genera within lesions may mean synergistic role with Treponema in hoof disease pathogenesis. Characterizing bacteria involved in lesion development, and their persistence during disease progression, provides evidence for science-based management decisions in TAHD infected elk populations.

SURVEILLANCE FOR AN EMERGENT HOOF DISEASE IN ELK (CERVUS ELAPHUS) IN THE US PACIFIC WEST SUPPLEMENTED BY 16S RRNA GENE AMPLICON SEQUENCING.

A novel hoof disease of elk (Cervus elaphus) was described in southwestern Washington, US, in 2008 and was subsequently diagnosed in an adjacent area in northwestern Oregon in 2014. The disease, currently referred to as treponeme-associated hoof disease (TAHD), is characterized by lesions ranging from mild erosions, to severe ulcers with underrunning of the hoof capsule and heel-sole junction, to overgrown and avulsed hoof capsules. Histologically, lesions exhibit epithelial erosion or ulceration, suppurative inflammation, and the presence of argyrophilic spirochetes. We used data collected by the Washington Department of Fish and Wildlife and Oregon Department of Fish and Wildlife from 2008 to 2017 as reference for disease distribution. We then conducted enhanced surveillance in 2018-20 by obtaining 164 submissions from four US Pacific West states. We detected TAHD for the first time in Idaho and northern California, as well as in multiple counties in Washington and Oregon where it had not been previously reported. Given the unexpectedly broad disease distribution, continued surveillance is warranted to determine the full geographic extent of TAHD. From samples of 22 elk, we investigated 16S rRNA gene amplicon sequencing as a technique that could be used to supplement TAHD surveillance. Operational taxonomic units of the family Spirochaetaceae were identified in 10 of 12 histologically diagnosed TAHD-positive cases and two of 10 TAHD-negative cases. Phyla Spirochaetae (P<0.008), Fusobacteria (P<0.006), and Tenericutes (P<0.01) were overrepresented in samples from TAHD-positive feet when compared with TAHD-negative elk. A unique spirochete, PT19, was detected in hooves of 11 elk and from at least one elk in each state. Results support the use of 16S rRNA gene amplicon sequencing as a reliable and informative tool to supplement investigations into distribution and etiology of this presumed polybacterial disease.

Lesion Material From Treponema-Associated Hoof Disease of Wild Elk Induces Disease Pathology in the Sheep Digital Dermatitis Model.

A hoof disease among wild elk (Cervus elaphus) in the western United States has been reported since 2008. Now present in Washington, Oregon, Idaho, and California, this hoof disease continues to spread among elk herds suggesting an infectious etiology. Causing severe lesions at the hoof-skin junction, lesions can penetrate the hoof-horn structure causing severe lameness, misshapen hooves, and in some cases, sloughed hooves leaving the elk prone to infection, malnutrition, and premature death. Isolated to the feet, this disease has been termed treponeme-associated hoof disease due to the numerous Treponema spp. found within lesions. In addition to the Treponema spp., treponeme-associated hoof disease shares many similarities with digital dermatitis of cattle and livestock including association with several groups of anaerobic bacteria such as Bacteroides, Clostridia, and Fusobacterium, neutrophilic inflammatory infiltrate, and restriction of the disease to the foot and hoof tissues. To determine if there was a transmissible infectious component to this disease syndrome, elk lesion homogenate was used in a sheep model of digital dermatitis. Ten animals were inoculated with lesion material and lesion development was followed over 7 weeks. Most inoculated feet developed moderate to severe lesions at 2- or 4-weeks post-inoculation timepoints, with 16 of 18 feet at 4 weeks also had spirochetes associated within the lesions. Histopathology demonstrated spirochetes at the invading edge of the lesions along with other hallmarks of elk hoof disease, neutrophilic inflammatory infiltrates, and keratinocyte erosion. Treponema-specific PCR demonstrated three phylotypes associated with elk hoof disease and digital dermatitis were present. Serum of infected sheep had increased anti-Treponema IgG when compared to negative control sheep and pre-exposure samples. Analysis of the bacterial microbiome by sequencing of the bacterial 16S rRNA gene showed a community structure in sheep lesions that was highly similar to the elk lesion homogenate used as inoculum. Bacteroidies, Fusobacterium, and Clostridia were among the bacterial taxa overrepresented in infected samples as compared to negative control samples. In conclusion, there is a highly transmissible, infectious bacterial component to elk treponeme-associated hoof disease which includes several species of Treponema as well as other bacteria previously associated with digital dermatitis.

Integrating Network Pharmacology and Molecular Docking to Analyse the Potential Mechanism of action of Macleaya cordata (Willd.) R. Br. in the Treatment of Bovine Hoof Disease.

Based on network pharmacological analysis and molecular docking techniques, the main components of M. cordata for the treatment of bovine relevant active compounds in M. cordata were searched for through previous research bases and literature databases, and then screened to identify candidate compounds based on physicochemical properties, pharmacokinetic parameters, bioavailability, and drug-like criteria. Target genes associated with hoof disease were obtained from the GeneCards database. Compound-target, compound-target-pathway-disease visualization networks, and protein-protein interaction (PPI) networks were constructed by Cytoscape. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed in R language. Molecular docking analysis was done using AutoDockTools. The visual network analysis showed that four active compounds, sanguinarine, chelerythrine, allocryptopine and protopine, were associated with the 10 target genes/proteins (SRC, MAPK3, MTOR, ESR1, PIK3CA, BCL2L1, JAK2, GSK3B, MAPK1, and AR) obtained from the screen. The enrichment analysis indicated that the cAMP, PI3K-Akt, and ErbB signaling pathways may be key signaling pathways in network pharmacology. The molecular docking results showed that sanguinarine, chelerythrine, allocryptopine, and protopine bound well to MAPK3 and JAK2. A comprehensive bioinformatics-based network topology strategy and molecular docking study has elucidated the multi-component synergistic mechanism of action of M. cordata in the treatment of bovine hoof disease, offering the possibility of developing M. cordata as a new source of drugs for hoof disease treatment.

Acute phase response and clinical manifestation in outbreaks of interdigital phlegmon in dairy herds.

Several Finnish dairy herds have suffered from outbreaks of interdigital phlegmon (IP). In these new types of outbreaks, morbidity was high and clinical signs severe, resulting in substantial economic losses for affected farms. In our study, we visited 18 free stall dairy herds experiencing an outbreak of IP and 3 control herds without a similar outbreak. From a total of 203 sampled cows, 60 suffered from acute stage IP. We demonstrated that acute phase response of bovine IP was evident and therefore an appropriate analgesic should be administered in the treatment of affected animals. The response was most apparent in herds with high morbidity in IP and with a bacterial infection comprising Fusobacterium necrophorum and Dichelobacter nodosus, indicating that combination of these two bacterial species affect the severity of the disease.

Animal board invited review: Dairy cow lameness expenditures, losses and total cost.

Lameness is one of the most costly dairy cow diseases, yet adoption of lameness prevention strategies remains low. Low lameness prevention adoption might be attributable to a lack of understanding regarding total lameness costs. In this review, we evaluated the contribution of different expenditures and losses to total lameness costs. Evaluated expenditures included labor for treatment, therapeutic supplies, lameness detection and lameness control and prevention. Evaluated losses included non-saleable milk, reduced milk production, reduced reproductive performance, increased animal death, increased animal culling, disease interrelationships, lameness recurrence and reduced animal welfare. The previous literature on total lameness cost estimates was also summarized. The reviewed studies indicated that previous estimates of total lameness costs are variable and inconsistent in the expenditures and losses they include. Many of the identified expenditure and loss categories require further research to accurately include in total lameness cost estimates. Future research should focus on identifying costs associated with specific lameness conditions, differing lameness severity levels, and differing stages of lactation at onset of lameness to provide better total lameness cost estimates that can be useful for decision making at both the herd and individual cow level.

A Review of the Design and Management of Footbaths for Dairy Cattle.

This article summarizes current footbath practices, questions the mechanism by which footbaths function, and reviews the available scientific literature testing footbaths in the field. Copper sulfate appears the most efficacious agent to include in a footbath program, but disposal concerns should limit the frequency of its use. Other agents such as formaldehyde have some merit when used with care. Use of water alone in a flush bath appears to have minimal impact. Footbaths should be used as infrequently as possible to achieve lameness prevention goals for the herd.

[Influence of lameness caused by hoof disease on welfare in sheep and goats. A comment on the guidelines of the German Small Ruminant Veterinary Association]. / Der Einfluss von Klauenerkrankungen auf das Tierwohl von Schaf und Ziege. Ein Kommentar zu den Haltungsempfehlungen der DVG.

The publication of the "Recommendations of husbandry and welfare of sheep and goats" by the German Small Ruminant Veterinary Association provides guidelines that are also used for assessing animal welfare. The included statements concerning lameness are reviewed according to the available literature and commented on with the help of practical examples. Diagnostic tools, criteria, appropriate measures and target values are discussed to facilitate the application of the guidelines.