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

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

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

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

Mendelian randomization analysis of 34,497 German Holstein cows to infer causal associations between milk production and health traits.

BACKGROUND: Claw diseases and mastitis represent the most important health issues in dairy cattle with a frequently mentioned connection to milk production. Although many studies have aimed at investigating this connection in more detail by estimating genetic correlations, they do not provide information about causality. An alternative is to carry out Mendelian randomization (MR) studies using genetic variants to investigate the effect of an exposure on an outcome trait mediated by genetic variants. No study has yet investigated the causal association of milk yield (MY) with health traits in dairy cattle. Hence, we performed a MR analysis of MY and seven health traits using imputed whole-genome sequence data from 34,497 German Holstein cows. We applied a method that uses summary statistics and removes horizontal pleiotropic variants (having an effect on both traits), which improves the power and unbiasedness of MR studies. In addition, genetic correlations between MY and each health trait were estimated to compare them with the estimates of causal effects that we expected. RESULTS: All genetic correlations between MY and each health trait were negative, ranging from - 0.303 (mastitis) to - 0.019 (digital dermatitis), which indicates a reduced health status as MY increases. The only non-significant correlation was between MY and digital dermatitis. In addition, each causal association was negative, ranging from - 0.131 (mastitis) to - 0.034 (laminitis), but the number of significant associations was reduced to five nominal and two experiment-wide significant results. The latter were between MY and mastitis and between MY and digital phlegmon. Horizontal pleiotropic variants were identified for mastitis, digital dermatitis and digital phlegmon. They were located within or nearby variants that were previously reported to have a horizontal pleiotropic effect, e.g., on milk production and somatic cell count. CONCLUSIONS: Our results confirm the known negative genetic connection between health traits and MY in dairy cattle. In addition, they provide new information about causality, which for example points to the negative energy balance mediating the connection between these traits. This knowledge helps to better understand whether the negative genetic correlation is based on pleiotropy, linkage between causal variants for both trait complexes, or indeed on a causal association.

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

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

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

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

Association between a genetic index for digital dermatitis resistance and the presence of digital dermatitis, heel horn erosion, and interdigital hyperplasia in Holstein cows.

Digital dermatitis (DD) is a polybacterial disease endemic to most UK dairy farms. It poses a major financial and welfare threat and is characterized by high incidence and recurrence rates. We aimed to investigate the association between the UK EBV for resistance to digital dermatitis, the digital dermatitis index (DDI), and the frequency of DD, heel horn erosion (HHE), and interdigital hyperplasia (IH) in a population of Holstein dairy cows. We enrolled and genotyped 2,352 cows from 4 farms in a prospective cohort study. Foot lesion records were recorded by veterinary surgeons for each animal at 4 time points during a production cycle, starting at approximately 2 mo before calving and ending in late lactation. Importantly, these records were not used in the calculation of the DDI. Lesion records were matched to the animal's own DDI (n = 2,101) and their sire's DDI (n = 1,812). Digital dermatitis index values in our study population ranged from -1.41 to +1.2 and were transformed to represent distance from the mean expressed in SD. The relationship between the DDI and the presence of DD was investigated using a logistic regression model, with farm, parity, and a farm-parity interaction fitted as covariates. A multivariable logistic regression model was fitted to evaluate the relationship between HHE and DDI with farm fitted as a covariate. Finally, a univariable logistic regression model with DDI as explanatory variable was used to investigate the relationship between IH and DDI. The odds ratio of an animal being affected by DD was 0.69 for 1 SD increase in the animal's DDI (95% CI = 0.63-0.76). The odds of HHE and IH were 0.69 (95% CI = 0.62-0.76) and 0.58 (95% CI = 0.49-0.68) respectively for 1 SD increase in DDI. The adjusted probability of DD was 32% (95% CI = 27-36%) for cows with mean DDI value of 0, while it was 24% (95% CI = 20-29%) in cows with a DDI value of +1. Sire DDI breeding values were standardized in the same way and then binned into terciles creating an ordinal variable representing bulls of high, medium, and low genetic merit for DD resistance. The daughters of low genetic merit bulls were at 2.05 (95% CI = 1.60-2.64), 1.96 (95% CI = 1.53-2.50), and 2.85 (95% CI = 1.64-5.16) times greater odds of being affected by DD, HHE, and IH, respectively, compared with the daughters of high genetic merit bulls. The results of this study highlight the potential of digital dermatitis genetic indexes to aid herd management of DD, and suggest that breeding for resistance to DD, alongside environmental and management control practices, could reduce the prevalence of the disease.

Genetic evaluations and genome-wide association studies for specific digital dermatitis diagnoses in dairy cows considering genotype × housing system interactions.

The present study aimed to use detailed phenotyping for the claw disorder digital dermatitis (DD) considering specific DD stages in 2 housing systems (conventional cubicle barns [CON] and compost-bedded pack barns [CBPB]) to infer possible genotype × housing system interactions. The DD stages included 2,980 observations for the 3 traits DD-sick, DD-acute, and DD-chronic from 1,311 Holstein-Friesian and 399 Fleckvieh-Simmental cows. Selection of the 5 CBPB and 5 CON herds was based on a specific protocol to achieve a high level of herd similarity with regard to climate, feeding, milking system, and location, but with pronounced housing-system differences. Five other farms had a "mixed system" with 2 subherds, one representing CBPB and the other one CON. The CBPB system was represented by 899 cows (1,530 observations), and 811 cows (1,450 observations) represented the CON system. The average disease prevalence was 20.47% for DD-sick, 13.88% for DD-acute, and 5.34% for DD-chronic, with a higher prevalence in CON than in CBPB. After quality control of 50K genotypes, 38,495 SNPs from 926 cows remained for the ongoing genomic analyses. Genetic parameters for DD-sick, DD-acute, and DD-chronic were estimated by applying single-step approaches for single-trait repeatability animal models considering the whole dataset, and separately for the CON and CBPB subsets. Genetic correlations between same DD traits from different housing systems, and between DD-sick, DD-chronic, and DD-acute, were estimated via bivariate animal models. Heritabilities based on the whole dataset were 0.16 for DD-sick, 0.14 for DD-acute, and 0.11 for DD-chronic. A slight increase of heritabilities and genetic variances was observed in CON compared with the "well-being" CBPB system, indicating a stronger genetic differentiation of diseases in a more challenging environment. Genetic correlations between same DD traits recorded in CON or CBPB were close to 0.80, disproving obvious genotype × housing system interactions. Genetic correlations among DD-sick, DD-acute and DD-chronic ranged from 0.58 to 0.81. SNP main effects and SNP × housing system interaction effects were estimated simultaneously via GWAS, considering only the phenotypes from genotyped cows. Ongoing annotations of potential candidate genes focused on chromosomal segments 100 kb upstream and downstream from the significantly associated candidate SNP. GWAS for main effects indicated heterogeneous Manhattan plots especially for DD-acute and DD-chronic, indicating particularities in disease pathogenesis. Nevertheless, a few shared annotated potential candidate genes, that is, METTL25, AFF3, PRKG1, and TENM4 for DD-sick and DD-acute, were identified. These genes have direct or indirect effects on disease resistance or immunology. For the SNP × housing system interaction, the annotated genes ASXL1 and NOL4L on BTA 13 were relevant for DD-sick and DD-acute. Overall, the very similar genetic parameters for the same traits in different environments and negligible genotype × housing system interactions indicate only minor effects on genetic evaluations for DD due to housing-system particularities.

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

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

Etiology and epidemiology of digital dermatitis in Australian dairy herds.

Bovine digital dermatitis (BDD) is an important cause of lameness in dairy cows worldwide. However, very little is known about this disease in Australian herds, which are predominantly managed on pasture. The primary objectives of this cross-sectional study were to describe the presence and prevalence of BDD in Australian dairy herds and to characterize the microbiota of healthy skin and M4 lesions of BDD-affected, pasture-managed cows. Cows from 71 dairy herds were examined at milking time to identify the presence of BDD lesions. True prevalence was estimated using Bayesian methods with informative priors for sensitivity and specificity. Biopsy samples (n = 60) were collected from cows with and without BDD lesions in 7 pasture-based herds. The microbiota in the superficial and deep strata of each tissue biopsy were characterized via sequencing of the V3-V4 region of the bacterial ribosomal RNA gene. Lesions were detected in 1,817 (11.5%) of 15,813 cows and in 68 of 71 (95.8%) herds. The median herd-level apparent and true prevalences of BDD were 8.5% and 18.1%, respectively, but prevalences varied considerably between farms. On farms with BDD, M4 lesions accounted for 70% to 100% of all lesions (interquartile range = 95.1%-100%, median = 100%); M2 lesions (i.e., large ulcerative lesions) were observed at low prevalence (<2.2%) in the few herds (7/71, 9.9%) where they were found. There was a significant difference in the composition of the microbiota between healthy skin and M4 lesions but not between superficial and deep tissue layers. Several gut- and effluent-associated bacterial taxa, including Lentimicrobium and Porphyromonas, which have previously been associated with BDD, were abundant in BDD lesions but not in control biopsies. Our study supports the idea that such taxa are involved in, although possibly not essential to, lesion development and persistence in pasture-managed cows in Australia. Our results also suggest that Dichelobacter may contribute to the disease process. We conclude that BDD is likely to occur in most Australian dairy farms, but that further studies are needed to identify its effect on cow welfare and productivity. Further investigation of the etiology of BDD in Australian dairy herds is also necessary to inform prevention and control strategies.

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.

Comparative efficacy of stannous fluoride and zinc sulfate solution to copper sulfate footbath solutions for the treatment and prevention of digital dermatitis in lactating dairy cows.

The objective of this study was to determine whether a novel footbath solution containing stannous fluoride (SnF2) was superior to 5% copper sulfate solution for the treatment and prevention of digital dermatitis (DD) in dairy cattle. Study 1 was conducted over 4 wk in Missouri and involved 34 lactating Holstein-Friesian cows with hind feet DD lesions. Cows in group SF walked through a footbath containing a proprietary formulation of SnF2 once weekly, whereas cows in group CS walked through a 5% CuSO4 footbath once daily for 5 d each week. Study 2 was conducted over 8 wk in California and involved 40 lactating Holstein-Friesian cows with hind feet DD lesions. Cows in group SF walked through a SnF2 footbath for 3 consecutive days and then once a week for the following 7 wk. Cows in group CS walked through a 5% CuSO4 footbath 3 times each week for 8 wk. Data collection included lesion type, lesion area, locomotion score, and pain score. Digital dermatitis was actively transmitted in study 1, and lesion area and locomotion scores were lower in group SF than group CS. In contrast, DD was not actively transmitted in study 2, and lesion area and locomotion scores were similar in groups SF and CS. Stannous fluoride delayed the development of active DD lesions in study 1 compared with copper sulfate, with a lower relative risk (0.57) of a hind foot developing an active DD lesion over 28 d. However, SnF2 decreased the rate that active DD lesions transitioned to M3, M4, or M0 lesions compared with 5% copper sulfate in both studies, with the relative risk of a hind foot with an active DD lesion transitioning to M3, M4, or M0 in group SF being slightly lower in study 1 (0.83) and study 2 (0.90) than in group CS. Our findings demonstrated that walking cows through a stannous fluoride footbath once per week in a herd undergoing active transmission of infection was more effective in preventing active DD lesions, but less effective in treating active DD lesions, than walking cows through a copper sulfate footbath 4 times per week. The novel SnF2 footbath solution shows promise for controlling DD in dairy herds that want an alternative footbath solution to CuSO4 and are interested in limiting the environmental accumulation of copper.

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

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

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

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

Dissecting the molecular diversity and commonality of bovine and human treponemes identifies key survival and adhesion mechanisms.

Here, we report the first complete genomes of three cultivable treponeme species from bovine digital dermatitis (DD) skin lesions, two comparative human treponemes, considered indistinguishable from bovine DD species, and a bovine gastrointestinal (GI) treponeme isolate. Key genomic differences between bovine and human treponemes implicate microbial mechanisms that enhance knowledge of how DD, a severe disease of ruminants, has emerged into a prolific, worldwide disease. Bovine DD treponemes have additional oxidative stress genes compared to nearest human-isolated relatives, suggesting better oxidative stress tolerance, and potentially explaining how bovine strains can colonize skin surfaces. Comparison of both bovine DD and GI treponemes as well as bovine pathogenic and human non-pathogenic saprophyte Treponema phagedenis strains indicates genes encoding a five-enzyme biosynthetic pathway for production of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, a rare di-N-acetylated mannuronic acid sugar, as important for pathogenesis. Bovine T. phagedenis strains further differed from human strains by having unique genetic clusters including components of a type IV secretion system and a phosphate utilisation system including phoU, a gene associated with osmotic stress survival. Proteomic analyses confirmed bovine derived T. phagedenis exhibits expression of PhoU but not the putative secretion system, whilst the novel mannuronic acid pathway was expressed in near entirety across the DD treponemes. Analysis of osmotic stress response in water identified a difference between bovine and human T. phagedenis with bovine strains exhibiting enhanced survival. This novel mechanism could enable a selective advantage, allowing environmental persistence and transmission of bovine T. phagedenis. Finally, we investigated putative outer membrane protein (OMP) ortholog families across the DD treponemes and identified several families as multi-specific adhesins capable of binding extra cellular matrix (ECM) components. One bovine pathogen specific adhesin ortholog family showed considerable serodiagnostic potential with the Treponema medium representative demonstrating considerable disease specificity (91.6%). This work has shed light on treponeme host adaptation and has identified candidate molecules for future diagnostics, vaccination and therapeutic intervention.

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.

A cross-sectional study of the prevalence of lameness and digital dermatitis in dairy cattle herds in Egypt.

BACKGROUND: Lameness is a significant problem for the dairy industry worldwide. No previous studies have evaluated the prevalence of lameness or digital dermatitis (DD) in dairy cattle herds in Egypt. A total of 16,098 dairy cows from 55 dairy herds in 11 Egyptian governorates underwent visual locomotion scoring using a 4-point scoring system. Cows that had a lameness score ≥ 2 were considered clinically lame. Following manure removal with water and using a flashlight, the cows' hind feet were examined in the milking parlour to identify DD lesions and classify with M-score. Furthermore, each cow was assigned a hock score (a 3-point scale) and a hygiene score (a 4-point scale). The cow-, within-and between-herd prevalence of lameness and DD and associated 95% confidence intervals (CI) were calculated. The prevalence of hock lesions and poor cow hygiene was also calculated. RESULTS: Of the examined cows, 6,883 were found to be clinically lame (42.8%, 95% CI = 42.0-43.5%). The average within-herd prevalence of lameness was 43.1% (95% CI = 35.9-50.3%). None of the dairy herds recruited into the study were found to be free from clinical lameness. The average within-herd prevalence of DD was 6.4% (95% CI = 4.9-8.0%). The herd-level prevalence of DD was 92.7% (95% CI = 85.9-99.6%). Active DD lesions (M1, M2, M4.1) were identified in 464 cows (2.9%) while inactive lesions (M3, M4) were identified in 559 cows (3.5%). The within-herd prevalence of hock lesions (score 2 or 3) was 12.6% (95% CI = 4.03-21.1%) while a severe hock lesion had within-herd prevalence of 0.31% (95% CI = 0.12-0.51%). Cow-level prevalence of hock lesions was 6.2% (n = 847, 95% CI = 5.8-6.2%). The majority of examined cows had a hygiene score of 4 (n = 10,814, prevalence = 70.3%, 95% CI = 69.5-71%). CONCLUSIONS: The prevalence of lameness was higher than prevalence estimates reported for other countries which could be due to differing management and/or environmental factors. DD was identified at lower prevalence in most herds but with high herd-level prevalence. Poor cow hygiene was notable in most herds. Measures to reduce the prevalence of lameness and to improve cow hygiene in dairy cattle herds in Egypt are therefore needed.

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.

Farm-level risk factors for digital dermatitis in dairy cows in mountainous regions.

Reduction of risk factors for bovine digital dermatitis (BDD) is crucial in current disease control. However, risk factors that might arise especially in mountainous regions are unknown until now, and an adapted BDD control program is consequently missing. The objective of this observational case-control study was to identify farm-level risk factors for BDD in dairy herds in mountainous regions. To investigate predictors for the occurrence of BDD, 100 farms were visited and information about herd characteristics and management practices, potentially relevant explanatory variables for either introduction or establishment of BDD, were gathered by completing a questionnaire with the farmer or herd manager. Within-herd prevalences of BDD assessed during 3 routine claw trimmings with an interval of 6 mo before the survey were used to define cases (BDD within-herd prevalence of ≥26% during each claw trimming) and controls (no BDD cases in each of the 3 routine claw trimmings before the survey). Data were analyzed using 2 separate binomial generalized linear models according to either establishment or introduction of BDD. After prescreening, 15 of 23 explanatory variables were included in the final analysis, which showed 3 variables related to introduction and establishment, each being significantly associated with the occurrence of BDD within a farm. Results of model 1 (i.e., aspects related to BDD introduction) revealed that access to mountain pastures during the summer season (odds ratio, 95% confidence interval: 0.12, 0.04-0.35), participation in dairy shows (0.32, 0.11-0.94), and the number of new animals introduced into the farm during the last 2 yr (1.28, 1.12-1.52) were significantly associated with the occurrence of BDD. Model 2 (i.e., aspects related to BDD establishment) showed that cows kept in freestalls were at higher risk for BDD compared with those kept in tiestalls (20.65, 1.59-649.37). Furthermore, number of days between diagnosis and treatment of a BDD lesion (10.31, 3.55-81.21) and the amount of concentrate feeding (median 5 kg) per cow and day (7.72, 2.46-6.47) were positively associated with BDD occurrence. In conclusion, the findings of this study provide a set of risk factors that are associated with BDD status within herds in mountainous regions. These results may help in development of adapted control programs for BDD in dairy cows.

Cost of lameness in dairy herds: An integrated bioeconomic modeling approach.

Foot disorders are costly health disorders in dairy farms, and their prevalence is related to several factors such as breed, nutrition, and farmer's management strategy. Very few modeling approaches have considered the dynamics of foot disorders and their interaction with farm management strategies within a holistic farm simulation model. The aim of this study was to estimate the cost of foot disorders in dairy herds by simulating strategies for managing lameness. A dynamic and stochastic simulation model (DairyHealthSim) was used to simulate the herd dynamics, reproduction management, and health events. A specific module was built for lameness and related herd-level management strategies. Foot disorder occurrences were simulated with a base risk for each etiology [digital dermatitis (DD), interdigital dermatitis, interdigital phlegmon, sole ulcer (SU), white line disease (WLD)]. Two state machines were implemented in the model: the first was related to the disease-induced lameness score (from 1 to 5), and the second concerned DD-state transitions. A total of 880 simulations were run to represent the combination of the following 5 scenarios: (1) housing (concrete vs. textured), (2) hygiene (2 different scraping frequencies), (3) the existence of preventive trimming, (4) different thresholds of DD prevalence detected and from which a collective footbath is applied to treat DD, and (5) farmer's ability to detect lameness (detection rate). Housing, hygiene, and trimming scenarios were associated with risk factors applied for each foot disorder etiologies. The footbath and lameness detection scenarios both determined the treatment setup and the policy of herd observance. The economic evaluation outcome was the gross margin per year. A linear regression model was run to estimate the cost per lame cow (lameness score ≥3), per case of DD and per week of a cow's medium lameness duration. The bioeconomic model reproduced a lameness prevalence varying from 26 to 98% depending on the management scenario, demonstrating a high capacity of the model to represent the diversity of the field situations. Digital dermatitis represented half of the total lameness cases, followed by interdigital dermatitis (28%), SU (19%), WLD (13%), and interdigital phlegmon (4%). The housing scenarios dramatically influenced the prevalence of SU and WLD, whereas scraping frequency and threshold for footbath application mainly determined the presence of DD. Interestingly, the results showed that preventive trimming allowed a better reduction in lameness prevalence than spending time on early detection. Scraping frequency was highly associated with DD occurrence, especially with a textured floor. The regression showed that costs were homogeneous (i.e., did not change with lameness prevalence; marginal cost equals average cost). A lame cow and a DD-affected cow cost €307.50 ± 8.40 (SD) and €391.80 ± 10.0 per year on average, respectively. The results also showed a cost of €12.10 ± 0.36 per week-cow lameness. The present estimation is the first to account for interactions between etiologies and for the complex DD dynamics with all the M-stage transitions, bringing a high level of accuracy to the results.

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.