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.
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BOVINE digital dermatitis (BDD) is an infectious condition primarily affecting the interdigital skin at the base of the claws. It was first reported in Italy (Cheli and Mortellaro 1974) and has since spread to most countries in the world, including the UK (Blowey and Sharp 1988). It is seen primarily in housed cattle in the Northern hemisphere and is much less common in the grazing systems adopted in Australia and New Zealand (Holzhauer and others 2006). The major aetiological infectious agents in BDD lesions are reported to be spirochaetal bacteria. In Germany, cloning of bacterial 16S rRNA genes identified five phylogroups of spirochaetes present within BDD lesions (Choi and others 1997). Three of these have since been isolated in the USA and UK, and are described as Treponema medium-like, Treponema phagedenis-like and Treponema denticola-like spirochaetes (Walker and others 1995, Stamm and others 2002, Evans and others 2008). Molecular epidemiological studies have subsequently identified high levels of association between these three treponeme phylogroups and BDD lesions in the UK and Germany (Nordhoff and others 2008, Evans and others 2009).
In the past 10 years, there have been a number of reports of ‘new’ disorders affecting the bovine digit. The first condition, termed ‘toe necrosis’ (TN), was clinically associated with herds with a high prevalence of BDD (Blowey 2008). It had been recognised clinically that, when BDD in herds was controlled, no further new cases of TN occurred, although existing lesions did not heal. The characteristic of this condition is the presence of a non-healing lesion penetrating through the horn capsule to involve the corium, typically at the wall of the toe. Longer-standing lesions lead to involvement of the tip of the pedal bone. A further two ‘non-healing’ hoof disorders initially present clinically as the classical hoof disorders white line disease (WLD) and sole ulcer (SU), but are typically more severe and are prefixed here with ‘non-healing’ to differentiate the conditions. The first, ‘non-healing white line disease’ (nhWLD), has been reported recently in the USA and Netherlands (Cook and Burgi 2008, Holzhauer 2008), and its presence in the UK is described here for the first time. The second disorder, ‘non-healing sole ulcer’ (nhSU), is also unusual, having been described only in the UK literature to date (Blowey 2008). In the opinion of one of these authors, these ‘non-healing’ manifestations are now appearing considerably more frequently than the original diseases.
Typical lesions of BDD involve only the digital and/or interdigital skin. This paper describes an association between BDD treponemes and these various ‘non-healing’ hoof disorders.
Materials and methods
The majority (n=38) of non-healing foot lesion samples included in this study were taken from ‘typical’ clinical cases, namely, from lame cows examined in the course of veterinary practice by one author (RWB) over a period of 18 months. A ‘typical case’ was defined as a non-healing lesion, often with excessive granulation tissue and with a characteristic pungent odour. The lesions were characterised as TN, nhWLD and nhSU. A sample of pungent tissue was taken during debridement of the lesion, with every effort being made to avoid environmental contamination. Many of the tissue samples consisted of a piece of hoof trimmed away using a hoof knife with a small fragment of soft tissue attached. For the tissue intended for bacterial culture, a small piece (5 mm) was separated and placed immediately in transport medium (Evans and others 2008). A further six non-healing samples were collected from animals in another region of the UK, using the same criteria. Nine samples representative of heel horn erosion, a disease of the hoof not involving complete horn penetration (in contrast to the non-healing manifestations), were also studied. Single hindfoot (control) hoof biopsies were also collected from the same location as the TN (n=4), nhWLD (n=7) and nhSU (n=5) manifestations from dairy cows (sent to slaughter) that did not have any evidence of BDD but were from BDD-endemic farms. Non-healing samples were stored overnight at 4°C or at −20°C (samples for PCR only) before being sent by post to the laboratory and then stored at −20°C on arrival. Heel horn erosion and control samples were transferred to the laboratory on ice and stored at −20°C.
Isolation and DNA extraction
Isolation of strains from non-healing lesions was carried out as previously described (Evans and others 2008). For extraction of genomic DNA from the non-healing lesions, control tissues and heel horn erosion samples, a DNeasy kit (Qiagen) was used in accordance with the manufacturer's instructions. Extracted genomic DNA was stored at −20°C.
All tissues were subjected to nested PCR assays (previously developed by the authors) specific for the three BDD treponeme groups, recently characterised and described by the authors' laboratory (Evans and others 2008, 2009). The initial PCR step used a universal bacterial primer pair encompassing the near complete 16S rRNA gene. The second/nested PCR step used primers encompassing smaller fragments (300 to 500 bp) within the 16S rRNA gene. All tissue extractions were subjected to a further Treponema genus-specific PCR test as described by Moore and others (2005). In order to validate each assay, water was used as a negative control; positive controls included genomic DNA from each of the three unique treponeme groups.
All the ‘non-healing’ hoof disorders observed in this study were particularly severe, penetrating the horn and involving the corium. The two non-healing disorders that have been recognised previously in classical forms (WLD and SU) were larger and much more destructive of the surrounding tissues than is usually seen. In total, 19 TN, 16 nhWLD and nine nhSU were observed and sampled. An example of each of these non-healing manifestations is shown in Fig 1. These non-healing hoof disorders had a characteristic pungent odour and a topical granular appearance, their response to standard treatments was poor and some eventually required amputation of the affected claw. All the cattle with lesions were from farms where BDD was endemic.
PCR of infected tissue
There were strong associations between the presence of BDD treponemes as assessed by PCR and each of the non-healing hoof disorders. The association of treponemes with TN is shown in Table 1, with DNA of Treponema medium-like, T phagedenis-like and T denticola-like BDD spirochaetes present in 94.7, 89.5 and 84.2 per cent of the samples, respectively. For nhWLD, the association with treponemes is shown in Table 2, with DNA of Treponema medium-like, T phagedenis-like and T denticola-like BDD spirochaetes present in 87.5, 93.8 and 87.5 per cent of the samples, respectively. The presence of BDD treponemes in nhSU samples is shown in Table 3, with DNA of Treponema medium-like, T phagedenis-like and T denticola-like BDD spirochaetes present in 77.8, 77.8 and 66.7 per cent of the samples, respectively. All three groups of BDD treponemes were present in 84.2, 81.3 and 55.6 per cent of TN, nhWLD and nhSU samples, respectively. The ‘general treponeme’ PCR was positive for all lesional hoof samples.
Typical heel horn erosion samples were also tested using the BDD treponeme PCR assays. There was no association between these typical heel horn erosion samples and the BDD treponemes, as shown in Table 4, although they were all positive by the general treponeme PCR.
PCR of healthy tissues
The results of PCR testing of the healthy horn samples from the different non-healing disease sites are shown in Table 4. Healthy tissues collected from animals without BDD lesions from farms endemic for BDD showed no presence of BDD-associated treponemes. The control tissues were positive by the general treponeme PCR in 68.8 per cent of samples.
Isolation was carried out for three representative samples of each non-healing disease. Spirochaetes were identified by phase contrast microscopy in all cases; unfortunately, however, viable cultures could not be maintained (due to contamination) despite the use of a previously successful culture technique involving the use of oral treponeme enrichment broth with fetal calf or rabbit serum supplement and rifampicin and enrofloxacin as selective agents, and using at least one round of subculture (Evans and others 2008).
In all the observed cases of TN, nhWLD and nhSU, the disorder included penetration through the horn capsule to involve the corium, resulting in exceptionally severe pain to the animal involved and, in some cases, extensive loss of the hoof horn. In addition to the obvious welfare implications of these non-healing disorders, there are significant economic effects such as reduction in milk yield and reproductive performance. In fact, as these disorders can require amputation of the affected claw, they will have even greater implications than classical BDD with regard to animal welfare and economic considerations, especially (as the authors believe) because these non-healing conditions are increasing in prevalence, in contrast to the classical manifestations of WLD and SU, which are not increasing.
For nhWLD, it has been reported that extensive debridement followed by a topical dressing of antibiotic and copper sulphate gave a reasonable response to treatment (Cook and Burgi 2008). No specific treatment trial was performed in the present study; however, the use of a topical antibiotic dressing plus parenteral long-acting (seven days) cephalosporin treatment by one of the authors (RWB) appeared to produce promising results in early lesions, and requires further investigation.
Given that these ‘non-healing’ lesions had a pungent odour and occurred frequently in herds with a high incidence of BDD, they were subjected to PCR assays to detect the BDD treponemes. These PCR assays have been previously used to report a high association between each of three unique, isolated BDD treponeme phylogroups and BDD lesions, and a co-association of 74.5 per cent (Evans and others 2009). In the present study, the BDD treponemes were highly associated with these non-healing manifestations, with an average association of 73.7 per cent for the three non-healing disease manifestations. In contrast, healthy control horn samples were PCR-negative for the BDD treponemes. These results suggest that BDD treponemes appear to be able to colonise other areas than those traditionally described, provided the presence of suitable conditions. Treponemes have already been reported as the likely cause of contagious ovine digital dermatitis (CODD) (Naylor and others 1998, Dhawi and others 2005, Sayers and others 2009) and more recently have been implicated in horse cankers (Moe and others 2010). Furthermore, it is worth noting that these new diseases show substantial similarity to CODD, where the corium is also frequently affected (Naylor and others 1998).
It is interesting that all three BDD treponeme groups were present in these non-healing hoof disorders, as is the case in BDD. Previous work carried out by the authors and others have suggested that BDD has a symbiotic treponemal pathogenesis (Klitgaard and others 2008, Nordhoff and others 2008, Evans and others 2009), and these non-healing foot manifestations would also appear to be representative of a similar symbiotic aetiology. The primary hoof disorders of WLD and SU are likely to be initiated by environmental trauma, whereas BDD is thought to be caused by bacteria alone, given the fact that the disease can be transmitted from animal to animal through lesional material (Read and Walker 1996). It is therefore interesting to speculate as to what has led to the involvement of the BDD treponemes in these foot disorders. The niche for each lesion is very similar to the typical BDD location, especially if trauma allows the horn integrity to be breached initially, thereby exposing the susceptible corium. Furthermore, it has been suggested that at least one of these groups of treponemes has the ability to downregulate the immune system (Zuerner and others 2007), and previous histological analyses of BDD lesions have provided evidence of an ineffective macrophage response to infection (Blowey and others 1994), making BDD treponemes ideal candidates for causing ‘non-healing’ lesions.
For comparative purposes, several heel horn erosion samples were tested with the BDD treponeme PCR assays. Heel horn erosion is representative of a disease that has not been reported to have a non-healing form and does not include penetration of the horn to the tissue below (Blowey 2008). There was no association between the BDD treponemes and heel horn erosion. This is consistent with a previous study that reported that heel horn erosion did not exhibit histological changes consistent with BDD (Blowey and Done 1995).
Interestingly, the authors found it more difficult (compared with the already difficult BDD lesions) to isolate treponemes from the non-healing lesions; unfortunately, it was not possible to obtain pure isolates. This might suggest that these manifestations may be strongly polymicrobial infections, although the lesions may just be representative of how much more contaminated these horn samples are compared with the skin lesions, because of continuous exposure to slurry.
The results from the ‘general treponeme’ PCR in this study are also interesting. It appears that treponemes other than those responsible for BDD appear to be commonly associated with the healthy horn of the foot; the authors have previously reported that these treponemes are also commonly present on the healthy interdigital cleft (Evans and others 2009) and healthy bovine udder tissue (Evans and others 2010). The treponemes, as a group, may be considered natural flora in a dairy farm environment. This could explain why they are commonly present on healthy hoof and foot skin while BDD treponemes are not always present. These non-BDD treponemes may represent reported rumen treponemes (Paster and Canale-Parola 1982) or may be commensal skin flora. A future study of interest may be to survey the feet of cattle at pasture to see whether they are also positive by the ‘general treponeme’ PCR.
This study has identified that these three types of foot disorders that affect dairy cows have strong associations with BDD treponemes. Further studies are required to determine whether any other specific bacteria are involved and to identify treatments for these disorders. Given the emergence of ‘non-healing’ presentations of foot lesions, even greater effort is required to reduce the incidence of BDD on farms.
This work was funded by a Biotechnology and Biological Sciences Research Council CEDFAS Research Grant (BBE0189201).
Provenance not commissioned; externally peer reviewed
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