Removal of bovine digital dermatitis-associated treponemes from hoof knives after foot-trimming: a disinfection field study.

BACKGROUND: Bovine digital dermatitis (BDD) is an infectious foot disease found commonly in dairy herds. Foot-trimming is an important husbandry procedure for reducing the ensuing lameness; however, epidemiological, and microbiological studies have identified this as a risk activity for transmitting BDD. Three disinfectants have previously been identified in laboratory work as effective for removing viable BDD-associated Treponema spp., from hoof knife blades. The present study enrolled 133 dairy cattle with BDD lesions, and swabbed hoof knife blades before and after foot-trimming, and after knife disinfection with one of three disinfectants (1:100 FAM30®, 2% Virkon® and 2% sodium hypochlorite) to assess their efficacy under field conditions. RESULTS: Detection of BDD treponeme phylogroup DNA was undertaken by direct PCR of swabs, and viable treponemes were detected by PCR of swab cultures after 6 weeks' incubation. Where hoof knives did not contact the lesion, BDD-associated treponemes were detected after foot-trimming in 12/22 (54.5%) cases by direct PCR and 1/22 (4.5%) cases by PCR of cultured organisms. Where contact was made with the lesion, 111/111 (100%) samples taken after trimming were positive by direct PCR and 47/118 (39.8%) were positive by culture PCR. Viable organisms were identified in cultures from lesion stages M2, M3, M4 and M4.1. No viable organisms were detected after disinfection of hoof knives. CONCLUSIONS: Hoof knives post-trimming were frequently contaminated with BDD-associated treponeme DNA. Viable organisms were identified in cultures whether contact had been made between hoof knife and lesion or not, although contact clearly increased the frequency of detection of viable organisms. The three disinfectants tested were effective for removing viable organisms. The disinfection protocol used in this study should therefore be considered reliable for adoption as standard industry practice.

Detection and Isolation of Digital Dermatitis Treponemes from Bovine Pressure Sores.

Pressure sores cause severe pain and discomfort in hospitalized people and in farmed cattle and are often infected with unknown bacteria. Pressure sores occur on the upper legs of 6-10% of recumbent cattle and are generally considered to be caused by constant pressure, commonly on bony areas of the limbs. This study analyzed pressure sores taken from the upper limbs of 14 cattle using isolation in culture and nested polymerase chain reaction (PCR) to detect treponemes associated with digital dermatitis (DD). A 100% association of DD treponemes with the pressure sores was demonstrated, but treponemes were shown not to be part of the normal skin microbiota. Immunohistochemistry showed an association of DD treponemes with lesions and particularly with the hair follicles in lesions, identifying the bacteria deep within wounds, thereby suggesting that they could contribute to lesion pathogenesis. The bacteria isolated from the pressure sore lesions were similar or identical on analysis of the 16S rRNA gene to those found in DD foot lesions in cattle, suggesting the same bacteria can infect multiple lesions. Indeed, the results of this study suggest that these spirochaetal bacteria may be expanding in host range and in their ability to colonize different tissues and contribute to a range of disease manifestations in farm animals.

Bovine ischaemic teat necrosis: a further potential role for digital dermatitis treponemes.

A recent outbreak of ischaemic teat necrosis (ITN) on mainland UK has resulted in large economic losses for dairy farmers. Typical cases start as an area of dry, thickened and encrusted skin on the medial aspect of the base of the teat, where the teat joins the udder, often with a fetid odour. The erosion spreads down the teat, often causing intense irritation, which in turn leads to more severely affected animals removing the entire teat. Due to the severity of ITN and the substantial economic costs to the industry, analyses were undertaken to ascertain if an infectious agent might be involved in the pathology. The study has considered a role for digital dermatitis (DD) treponemes in the aetiopathogenesis of ITN because, as well as being the prime bacteria associated with infectious lameness, they have been associated with a number of emerging skin diseases of cattle, including udder lesions. A high association between presence of DD-associated treponemes and incidence of ITN (19/22), compared with absence in the control population is reported. Furthermore, sequencing of the 16S rRNA gene of treponeme isolates supports the hypothesis that the identified treponemes are similar or identical to those isolated from classical foot DD lesions in cattle (and sheep). Further studies are required to allow effective targeted prevention measures and/or treatments to be developed.

A molecular epidemiology of treponemes in beef cattle digital dermatitis lesions and comparative analyses with sheep contagious ovine digital dermatitis and dairy cattle digital dermatitis lesions.

Bovine digital dermatitis (BDD) is an infective foot disease commonly reported in dairy cattle where Treponema are considered as the primary causative infectious agents. There still remains little definitive information on the etiology of BDD in beef cattle suggesting further investigations are warranted. Beef BDD lesions (n=34) and healthy beef foot tissues (n=38) were analysed by PCR for three BDD-associated Treponema phylogroups and also for Dichelobacter nodosus and Fusobacterium necrophorum. Spirochete culture was attempted on all BDD lesion samples. One or more BDD-associated Treponema phylogroups were detected in 100% of beef BDD lesions. "Treponema medium/Treponema vincentii-like", "Treponema phagedenis-like" and Treponema pedis spirochetes were identified in 27/34 (79%), 31/34 (91%) and 24/34 (71%) of BDD lesions, respectively. No BDD-associated treponeme DNA was amplified from beef healthy foot tissues. D. nodosus and F. necrophorum were present in 24/34 (71%) and 15/34 (44%) of lesions and 10/38 (26%) and 12/38 (32%) of healthy foot tissues, respectively. Twenty spirochetes were isolated from beef BDD lesions; 19 were representatives of the three BDD-associated Treponema phylogroups. One spirochete isolate shared less than 97% 16S rRNA gene similarity to the three cultivable BDD-associated Treponema phylogroups and therefore may represent a novel taxa of Treponema. Upon comparison, sheep contagious ovine digital dermatitis (CODD), dairy cattle and beef cattle BDD lesions appear to have extremely similar bacteriological data and therefore provides evidence of a shared etiopathogenesis posing concerns for cross-species transmission.

High-level association of bovine digital dermatitis Treponema spp. with contagious ovine digital dermatitis lesions and presence of Fusobacterium necrophorum and Dichelobacter nodosus.

Contagious ovine digital dermatitis (CODD) is an important foot disease in sheep, with significant animal welfare and economic implications. It is thought that CODD emerged from bovine digital dermatitis (BDD) via treponemal bacteria. With wildlife species such as elk now suffering a CODD-like disease, it is imperative to clarify these disease etiologies. A large investigation into treponemal association with CODD is warranted. CODD lesions (n = 58) and healthy sheep foot tissues (n = 56) were analyzed by PCR for the three BDD-associated Treponema phylogroups and two other lameness-associated bacteria, Dichelobacter nodosus and Fusobacterium necrophorum. Spirochete culture was also attempted on CODD lesions. "Treponema medium/Treponema vincentii-like," "Treponema phagedenis-like," and Treponema pedis spirochetes were identified in 39/58 (67%), 49/58 (85%), and 41/58 (71%) of CODD lesions, respectively. One or more BDD-associated Treponema phylogroups were detected in 100% of CODD lesions. Healthy foot tissues did not amplify BDD-associated Treponema phylogroup DNA. D. nodosus and F. necrophorum were present in 34/58 (59%) and 41/58 (71%) of CODD lesions and 22/56 (39%) and 5/56 (9%) of healthy foot tissues, respectively. Thirty-two spirochetes were isolated from CODD lesions, with representatives clustering with, and indistinguishable from, each of the three BDD-associated Treponema phylogroups based on 16S rRNA gene comparisons. This study for the first time demonstrates a high-level association for BDD treponeme phylogroups in CODD and their absence from healthy tissues, supporting the hypothesis that BDD treponemes play a primary causative role in CODD and confirming that the specific PCR assays are an effective differential diagnostic tool for CODD.

Isolation of digital dermatitis treponemes from hoof lesions in Wild North American Elk (Cervus elaphus) in Washington State, USA.

Since 2008, a large increase in the numbers of cases of lameness have been seen in wild North American elk (Cervus elaphus) from Washington State, USA. The most recent cases manifested as foot lesions similar both clinically and pathologically to those seen in digital dermatitis (DD) in cattle and sheep, a disease with a bacterial etiopathogenesis. To determine whether the same bacteria considered responsible for DD are associated with elk lameness, lesion samples were subjected to bacterial isolation studies and PCR assays for three phylogroups of relevant DD treponemes. The DD treponemes were isolated from lesional tissues but not from control feet or other areas of the diseased foot (including the coronary band or interdigital space), suggesting that the bacteria are strongly associated with DD lesions and may therefore be causal. In addition, PCR analysis revealed that all three unique DD treponeme phylotypes were found in elk hoof disease, and in 23% of samples, all 3 DD-associated treponemes were present in lesions. Sequence analysis of the 16S rRNA gene showed that the elk lesion treponemes were phylogenetically almost identical to those isolated from cattle and sheep DD lesions. The isolates were particularly similar to two of the three culturable DD treponeme phylotypes: specifically, the Treponema medium/Treponema vincentii-like and Treponema phagedenis-like DD spirochetes. The third treponeme culturable phylogroup (Treponema pedis), although detected by PCR, was not isolated. This is the first report describing isolation of DD treponemes from a wildlife host, suggesting that the disease may be evolving to include a wider spectrum of cloven-hoofed animals.

Presence of digital dermatitis treponemes on cattle and sheep hoof trimming equipment.

Digital dermatitis (DD) is an infectious foot disease causing severe lameness in dairy cattle (worldwide) and sheep (UK). This study investigated whether DD Treponema phylogroups can be present on equipment used to trim ruminant hooves and, therefore, consider this trimming equipment as a possible vector for the transmission of DD. Equipment was tested after being used to trim DD symptomatic and asymptomatic cattle and sheep hooves, and subsequently after disinfection of equipment. After trimming, 'Treponema medium/Treponema vincentii-like', 'Treponema phagedenis-like' and 'Treponema denticola/T putidum-like' DD spirochaetes, were shown to be present on 23/37 (62%), 21/37 (57%) and 20/37 (54%) of knives, respectively. After disinfection, detection rates for the DD treponemes were 9/37 (24%), 6/37 (16%) and 3/37 (8%), respectively. Following culture of a swab, an isolate belonging to the T phagedenis-like spirochaetes was identified from a knife sample after trimming a DD positive cow. No isolates were obtained from knife samples after disinfection. This new data has, for the first time, identified treponemes in the farm environment, and highlighted disinfection of hoof trimming equipment between animals and between farms, as a logical precaution to limit the spread of DD.

Identifying host pathogenic pathways in bovine digital dermatitis by RNA-Seq analysis.

Digital dermatitis is a painful foot disease compromising welfare in dairy cattle. The disease has a complex multibacterial aetiology, but little is known about its pathogenesis. In this study, gene expression in skin biopsies from five bovine digital dermatitis lesions and five healthy bovine feet was compared using RNA-Seq technology. Differential gene expression was determined after mapping transcripts to the Btau 4.0 genome. Pathway analysis identified gene networks involving differentially expressed transcripts. Bovine digital dermatitis lesions had increased expression of mRNA for α2-macroglobulin-like 1, a protein potentially involved in bacterial immune evasion and bacterial survival. There was increased expression of keratin 6A and interleukin 1ß mRNA in bovine digital dermatitis lesions, but reduced expression of most other keratin and keratin-associated genes. There was little evidence of local immune reactions to the bacterial infection present in lesions.

Association between bovine digital dermatitis treponemes and a range of 'non-healing' bovine hoof disorders.

This study describes the association between bovine digital dermatitis (BDD) treponemes and three 'non-healing' bovine hoof horn lesions, namely, 'toe necrosis' (TN), 'non-healing white line disease' (nhWLD) and 'non-healing sole ulcer' (nhSU), which are disorders that involve penetration through the horn capsule to involve the corium. In this study, these non-healing disorders (n=44) were identified as foot lesions that exhibited a topical granular appearance, exuded a typical pungent smell, were severely painful to the animal involved, and typically originated from farms where BDD is endemic. Given the similarities between these 'non-healing' lesions and BDD, the authors subjected samples of diseased tissue to PCR assays to detect the presence of DNA of BDD treponemes. All the three characterised BDD treponeme groups were identified as present together in 84.2, 81.3 and 55.6 per cent of samples of TN (n=19), nhWLD (n=16) and nhSU (n=9), respectively. In contrast, healthy control horn samples from similar sites (n=16) were PCR-negative for the BDD treponemes. Hence, these non-healing hoof lesions were strongly associated with BDD treponemes. Samples from typical heel horn erosions (n=9) were also subjected to BDD treponeme PCR assays and no association could be identified between the BDD treponemes and this horn manifestation.

Risk factors for increased rates of sole ulcers, white line disease, and digital dermatitis in dairy cattle from twenty-seven farms in England and Wales.

Claw lesion treatment records were recorded by farmers on 27 dairy farms (3,074 cows, 36,432 records) in England and Wales between February 2003 and February 2004. These were combined with farm environment and management data collected using a combination of direct observations, interviews with farmers, and milk recording data. Multilevel models were constructed for the 3 most frequently reported lesions related to lameness, namely, sole ulcers, white line disease, and digital dermatitis. Risks associated with an increased incidence of sole ulcers were parity 4 or greater, the use of roads or concrete cow tracks between the parlor and grazing, the use of lime on free stalls, and housing in free stalls with sparse bedding for 4 mo or more. The risks for white line disease were increasing parity and increasing herd size, cows at pasture by day and housed at night, and solid grooved concrete floors in yards or alleys. Solid grooved flooring was also associated with an increased risk of digital dermatitis, and cows 6 or more months after calving had a decreased risk of a first case of digital dermatitis. These results improve our understanding of the specific risks for 3 important lesions associated with bovine lameness and could be used as interventions in future clinical studies targeted at the reduction of specific lesions.

Associations between sole ulcer, white line disease and digital dermatitis and the milk yield of 1824 dairy cows on 30 dairy cow farms in England and Wales from February 2003-November 2004.

The milk yields of 1824 cows were used to investigate the effect of lesion-specific causes of lameness, based on farmer treatment and diagnosis of lame cows, on milk yield. A three-level hierarchical model of repeated test day yields within cows within herds was used to investigate the impact of lesion-specific causes of lameness (sole ulcer, white line disease, digital dermatitis and other causes) on milk yield before and after treatment compared with unaffected cows. Cattle which developed sole ulcer (SU) and white line disease (WLD) were higher yielding cattle before they were diagnosed. Their milk production fell to below that of the mean of unaffected cows before diagnosis and remained low after diagnosis. In cattle which developed digital dermatitis (DD) there was no significant difference in milk yield before treatment and a slightly raised milk yield immediately after treatment. The estimated milk loss attributable to SU and WLD was approximately 570 and 370 kg, respectively. These results highlight that specific types of lameness vary by herds and within herds they are associated with higher yielding cattle. Consequently lesion-specific lameness reduction programmes targeting the cow and farm specific causes of lameness might be more effective than generic recommendations. They also highlight the importance of milk loss when estimating the economic impact of SU and WLD on the farms profitability.

Management factors associated with impaired locomotion in dairy cows in England and Wales.

Forty-nine farms in England and Wales were visited on 4 occasions between February 2003 and March 2004. A total of 21,693 scores of locomotion were assigned to 7,722 cattle. Locomotion was assessed on a 3-point scale by observing the posture of a cow's back while standing and walking (1 = sound, 2 = not sound, 3 = lame). Data on measurable factors potentially associated with locomotion were collected from all farms using direct observations of the farm environment and a comprehensive farmer interview. The mean herd locomotion score was 1.77 +/- 0.02. There was no significant difference in mean herd locomotion scores between 5 herds housed in straw yards (1.72 +/- 0.02) and 44 herds housed in free stalls (1.78 +/- 0.02), possibly because of lack of power. A GLM was produced using data from the 44 herds housed in free stalls, with the mean farm locomotion score of all cows examined on all 4 visits as the outcome variable. Factors associated with an elevated locomotion score were dry cows kept in straw yards compared with free stalls (increase in locomotion score = 0.06 +/- 0.03), pregnant heifers kept with milking cows in winter compared with being kept with dry cows (increase in locomotion score = 0.09 +/- 0.03), aisle widths of < 3 m compared with widths of > or = 3 m (increase in locomotion score = 0.06 +/- 0.02), a curb height of < or = 15 cm compared with a height of > 15 cm (increase in locomotion score = 0.07 +/- 0.03), routine trimming of hooves of all cows by a hoof trimmer or by the farmer compared with no routine hoof trimming (increase in locomotion score = 0.18 +/- 0.04 and 0.13 +/- 0.03 respectively), feeding corn silage to milking cows compared with feeding other forage types (increase in locomotion score = 0.10 +/- 0.03), and the use of automatic scrapers in the free-stall barn compared with tractor scrapers (increase in locomotion score = 0.10 +/- 0.03). These variables were correlated with many other management variables. The use of automatic scrapers was correlated with the use of sawdust on rubber mats in free stalls. Curb height was negatively correlated with increasing distance of the neck rail from the front (head end) of the free stall. These putative risk factors support the hypothesis that locomotion score is linked to management factors; in particular, the combination of sawdust on rubber mats with automatic scrapers was associated with elevated locomotion scores.

Risk factors for reduced locomotion in dairy cattle on nineteen farms in The Netherlands.

A 3-point locomotion scoring system was used that incorporated the position of the back of cows while standing and when walking to investigate risk factors for elevated locomotion scores of 1,450 dairy cows on 19 farms in The Netherlands. Each of the farms was visited twice in an 18-mo period from February 2003 to July 2004. At each visit, all milking and dry cows were scored for locomotion by a single observer. Two multivariable regression models were constructed to identify factors associated with elevated mean locomotion score (increased abnormality) and the percentage of cows with the highest score (score 3). Risk factors for increased locomotion score were having a hoof-trimming stall with foot-lifting apparatus compared with not having such apparatus (increase in locomotion score = 0.15), presence of a footbath at the parlor exit or other site compared with not having a footbath on the farm (increase in locomotion score = 0.17 and 0.19, respectively), not providing supplemental vitamins and minerals to lactating cows compared with supplementing animals (increase in locomotion score = 0.17) and feeding corn silage to heifers compared with not doing so (increase in locomotion score = 0.10). The results provide a framework for hypotheses for future investigations of risk factors for high locomotion scores.

Testing white line strength in the dairy cow.

The tensile strength of 576 pieces of white line horn collected over 6 mo from 14 dairy cows restricted to parity 1 or 2 was tested. None of the cows had ever been lame. Seven cows were randomly assigned to receive 20 mg/d biotin supplementation, and 7 were not supplemented. Hoof horn samples were taken from zones 2 and 3 (the more proximal and distal sites of the abaxial white line) of the medial and lateral claws of both hind feet on d 1 and on 5 further occasions over 6 mo. The samples were analyzed at 100% water saturation. Hoof slivers were notched to ensure that tensile strength was measured specifically across the white line region. The tensile stress at failure was measured in MPa and was adjusted for the cross-sectional area of the notch site. Data were analyzed in a multilevel model, which accounted for the repeated measures within cows. All other variables were entered as fixed effects. In the final model, there was considerable variation in strength over time. Tensile strength was significantly higher in medial compared with lateral claws, and zone 2 was significantly stronger than zone 3. Where the white line was visibly damaged the tensile strength was low. Biotin supplementation did not affect the tensile strength of the white line. Results of this study indicate that damage to the white line impairs its tensile strength and that in horn with no visible abnormality the white line is weaker in the lateral hind claw than the medial and in zone 3 compared with zone 2. The biomechanical strength was lowest at zone 3 of the lateral hind claw, which is the most common site of white line disease lameness in cattle.