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Paper Digital dermatitis treponemes associated with a severe foot disease in dairy goats L. E. Sullivan, N. J. Evans, S. R. Clegg, S. D. Carter, J. E. Horsfield, D. Grove- White, J. S. Duncan A UK dairy goat herd was assessed after reports of a severe lameness problem of unknown aetiology. A lameness prevalence estimate was produced and individual clinical examination of 15 randomly selected lame goats was performed. Fifteen animals had foot lesions closely resembling contagious ovine digital dermatitis (CODD) in sheep. Eight of the goats examined presented with typical CODD lesions and seven showed what appeared to be a more severe CODD with under-running of the sole. Ten biopsy samples were obtained from the foot lesions and tested by PCR for the three previously isolated digital dermatitis (DD) Treponema phylogroups and culture of treponemes was attempted. Ninety per cent of the biopsy samples were positive for Treponema medium/Treponema vincentii-like spirochaetes and Treponema phagedenis-like DD spirochaetes and 80per cent were positive for Treponema pedis. Spirochaetes were successfully isolated from 50 per cent of lesion samples. Three isolates were identified as belonging to the T. phagedenis-like spirochaetes and two were identified as T. pedis. The frequent isolation of similar treponemes to those isolated from bovine digital dermatitis and CODD lesions and the identification of these DD-associated phylotypes in the vast majority of lesions support the hypothesis that this novel foot condition is associated with infection by DD treponemes, and given the similarities to CODD, it suggests a causal role. Introduction Lameness in dairy cattle and sheep has serious animal welfare and economic implications (Marshall and others 1991, Enting and others 1997, Hernandez and others 2001, Warnick and others 2001). Lameness is also reported in goats, although with fewer published studies. Prevalence data for lameness in goat herds and diagnosis of causes of lameness have been reported from several countries. In France, Mazurek and others (2007) reported that out of 108 goats, 12.5 per cent were lame and Christodoulopoulos (2009) identified a prevalence of 24 per cent in a herd of 170 goats in Greece. In the UK, Hill and others (1997) found an average lameness prevalence of 9.1 per cent (range 2.7–23.4 per cent) on four goat farms. In a study of 24 dairy goat farms in England and Wales, the mean lameness prevalence was 19.2 per cent (IQR 7.7–30.2 per cent). One of the goat herds visited Veterinary Record (2014) L. E. Sullivan, N. J. Evans, S. R. Clegg, S. D. Carter, Department of Infection Biology, Institute of Infection and Global Health, School of Veterinary Science, University of Liverpool, Science Park ic2, 146 Brownlow Hill, Liverpool, Merseyside L3 5RF, UK J. E. Horsfield, Lanes Veterinary Group, Green Lane Veterinary Centre, Leachfield Industrial Estate, Green Lane West, Cabus,
doi: 10.1136/vr.102858 Garstang PR3 1PR, UK D. Grove- White, J. S. Duncan, Department of Epidemiology and Population Health, Institute of Infection and Global Health, University of Liverpool, Leahurst Campus, Neston, Wirral CH64 7TE, UK E-mail for correspondence: [email protected] Provenance: not commissioned; externally peer reviewed Accepted November 3, 2014
had a lameness prevalence of 52.5 per cent, while three of the herds had no detectable lameness at the time of the visit (Anzuino and others 2010). More recently, similarly high levels of lameness (40 per cent and 67 per cent) were observed among dairy goats on two UK goat farms (Groenevelt and others 2013). Foot disorders recognised in goats are similar to those described in sheep (Winter 2011) and include: interdigital dermatitis, footrot (Duran and others 1990), heel horn erosion (Christodoulopoulos 2009), white line disease, foreign body penetrations (Mgasa and Arnbjerg, 1993) and overgrown feet (Hill and others 1997). In the study by Groenevelt and others (2013), laminitis accompanied by infection with Treponema bacteria was identified as the cause of lameness in two dairy goat herds. Contagious ovine digital dermatitis (CODD) is an emerging severe foot disease of sheep which was first reported in the UK in 1997 (Harwood and others 1997, Davies and others 1999). It has been shown to be associated with Treponema bacteria (Sayers and others 2009) and is hypothesised to have derived from digital dermatitis (DD) lesions in dairy cows (Dhawi and others 2005). CODD is now widespread in UK sheep (Kaler and Green 2008, Angell and others 2014). Although goats and sheep share many foot diseases, CODD is yet to be reported in goats; however, having possibly crossed into sheep from one host species, there was always a chance that this could happen again and infection of other domesticated species could occur. Here, the authors report the clinical and microbiological findings of a severe lameness problem in a UK dairy goat herd. Clinical descriptions of lesions were produced and lesion biopsies were investigated by PCR and bacterial culture for an association with the same Treponema bacteria found in bovine digital dermatitis (BDD) and CODD. March 14, 2015 | Veterinary Record
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Paper Materials and methods Farm details and clinical description of lesions Following reports of a severe lameness problem of unknown aetiology, a UK goat herd was assessed by the attending veterinary surgeons ( JSD and JEH) in October 2013. The herd consisted of 1000 milking goats of Anglo-Nubian and British Saanen breed. The goats were housed year round in four straw yards (250 goats per yard), which were cleaned out every six to eight weeks. Milking occurred twice a day and goats were fed a ration of grass silage and cereal-based concentrate. Infection with caprine arthritis encephalitis virus had previously been diagnosed on the farm. All goats were routinely foot trimmed every three months and the herd walked through a copper sulphate footbath post milking, twice weekly. The farmer estimated that 30 per cent of the herd were lame and his current treatment for lame goats was foot trimming and topical application of oxytetracycline spray; in some but not all lameness cases the goats would also be treated with a course of the parental antibiotic, ceftiofur (Naxcel, Zoetis). However, it appeared this was not controlling the disease effectively and a high level of lameness was apparent on the farm for several months. The prevalence of lameness in this herd was estimated by the attending veterinary surgeon after lameness scoring the entire herd on exit from the milking parlour. Due to the rate and numbers of goats exiting the parlour, a simple lame/not lame scoring system was used (Phythian and others 2013). On this basis, 65 per cent of the herd were identified as lame. There appeared to be no observable difference in lameness prevalence between the four straw yards. Fifteen lame goats were randomly selected for further examination. All were non-weight bearing and were affected on one leg only. Eight of the 15 lame goats had foot lesions showing very close resemblance to the typical presentation of CODD in sheep. These goats displayed separation of the hoof capsule at the level of the coronary band with the underlying exposed tissue appearing haemorrhagic and granulomatous (Fig 1a, b). These lesions appeared to originate at the coronary band. The lesions on the other seven goats examined demonstrated an even more severe presentation, with loss of solar horn accompanied by marked granulation and haemorrhage of the sole (Fig 1c). The lesions on these seven goats therefore presented lesions similar to CODD, but the origin of the lesions was indistinguishable due to their severity.
Sample collection Ten of the goats with foot lesions were chosen randomly from the goats examined and the lesions were biopsied using a 3 mm punch biopsy under local anaesthesia (Evans and others 2008). Lesion samples were divided into two with half transferred into transport medium and placed on ice for subsequent Treponema culture. Transport medium consisted of oral treponeme enrichment broth (OTEB; Anaerobe Systems, Morgan Hill, California, USA) and the antibiotics rifampicin (5 μg/ml) and enrofloxacin (5 μg/ml). The remaining tissue from lesions (for PCR analysis) was transported on ice and stored at −20° C. Healthy goat foot tissues (n=10) were used to serve as negative controls. Single rear-hoof skin biopsies were collected from each hindfoot of five dairy goats sent to slaughter that did not have any evidence of foot lesions. All healthy tissue samples were transported and stored as above.
DNA extraction and treponeme PCR assays For PCR analysis, frozen tissues from the lesions and healthy tissues were thawed and DNA extracted using a DNeasy Kit (Qiagen, UK) according to the manufacturer’s instructions, and genomic DNA was stored at −20° C. Samples were subjected to nested PCR assays specific for the three DD-associated treponeme groups, “Treponema medium/Treponema vincentii-like”, “Treponema phagedenis-like” and Treponema pedis described by Evans and others (2008, 2009) with resulting PCR products Veterinary Record | March 14, 2015
encompassing 300–500 bp of the 16S rRNA gene. All foot samples were also subjected to the Treponema genus PCR assay (Moore and others 2005).
Culture of spirochaetes Bacterial isolation was attempted on all lesion samples (n=10). A (1–1.5 mm) piece of tissue was transferred into an anaerobic cabinet (85 per cent N2, 10 per cent H2 and 5 per cent CO2, 36° C). Each was inoculated into OTEB (Anaerobe Systems) with 10 per cent fetal calf serum (FCS) and the antibiotics rifampicin (5 μg/ml) and enrofloxacin (5 μg/ml). After two to five days, bacteria were then subcultured on fastidious anaerobe agar plates (LabM, Bury, UK) with 5 per cent defibrinated sheep blood, 10 per cent FCS and antibiotics as above. After one to two weeks, single colonies were inoculated into growth media and were checked for pure culture by phase contrast microscopy. DNA was extracted from treponeme cultures and the isolated organisms were identified using a 16S rRNA gene PCR and gene sequencing as previously described (Evans and others 2008). Bacterial isolation was not attempted on healthy tissues following treponeme PCR assay results.
Phylogenetic analysis of spirochaete isolates To understand the relationship of the isolated spirochaetes with other treponemes, a phylogenetic tree was produced from the aligned and trimmed 16S rRNA gene PCR product sequences from the isolates and relevant microorganisms are available in GenBank. Sequences were assembled into a double-stranded consensus sequence using Chromas Pro 1.41 (Technelysium Pty Ltd). Consensus sequences were aligned by Clustal W and trimmed using Mega 5.2 (Tamura and others 2011). For tree analysis, the most appropriate evolution model was predicted using ‘model test’ as implemented in the TOPALi program(Milne and others 2009). The final model for nucleotide substitutions chosen was the general time reversal model (Tavare 1986), used to infer a bootstrapped maximum likelihood tree; bootstrapping was performed 10, 000 times.
Results Treponeme PCR assays The 10 biopsy samples obtained from the foot lesions were all positive for the Treponema genus-specific PCR (Table 1, samples 1–10), which detects all Treponema species, including pathogens and commensals. The DD group-specific PCR assays found T. medium/T. vincentii-like spirochaetes and T. phagedenis-like DD spirochaetes in 90 per cent of samples and 80 per cent were positive for T. pedis. All lesional samples were positive for at least one and usually more of the DD-associated Treponema phylogroups upon PCR analysis (Table 1). All healthy foot tissues were negative for all three of the DD group-specific PCR assays. Of the 10 healthy foot tissue samples, seven were positive for the Treponema genus-specific PCR.
Isolation of spirochaetes Spirochaetes were successfully isolated from 50 per cent of cultured lesion samples (n=10) (Table 1). The isolates G6JD, G7JD and G10JD (GenBank accession codes: KJ206529, KJ206530, KJ206532, respectively) were identified as belonging to the “T. phagedenis-like” spirochaetes and shared 100 per cent 16S rRNA gene sequence identity with the “T. phagedenis-like” DD spirochaete strain T320A (GenBank accession: EF061261), previously isolated from a dairy cow DD lesion in the UK (Evans and others 2008). Isolates G2JD and G9JD (GenBank accession codes: KJ206528, KJ206531, respectively) belonged to the T. pedis spirochaetes and shared 99 per cent 16S rRNA gene sequence identity with T. pedis T3552B (NR 044064) previously isolated from a dairy cow DD lesion in the UK (Evans and others 2008).
Phylogenetic analysis of isolated spirochaetes All successfully cultured isolates were clustered with their respective closest spirochaete relatives upon phylogenetic
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FIG 1: (a) “CODD type lesion” in a dairy goat. (b) “CODD type lesion” in a goat with separated hoof horn removed to show underlying granulomatous and haemorrhagic tissue. (c) Goat foot with a more severe “CODD type lesion” affecting sole of foot.
analysis (Fig 2). Goat isolates from samples 6, 7 and 10 were clustered closely with the “T. phagedenis-like” spirochaetes, as would be expected from the PCR analyses of the lesional material. Samples 2 and 9 were clustered with the T. pedis spirochaetes; however, they formed a separate clade likely due to five nucleotide substitutions in the 16S rRNA gene (A69G, T73C, A219G, T405C, A440G).
Discussion The foot disease documented here was extremely severe with no early lesions identified; although in the majority of cases it appeared that the lesions originated at the coronary band, as with CODD in sheep (Davies and others 1999, Winter 2008). The lesions in eight of the goats examined (Fig 1a, b), where there was separation of hoof horn at the level of the coronary band accompanied by extensive under-running of the hoof horn capsule, bear a marked similarity to the published lesion descriptions of CODD in sheep (Davies and others 1999, Winter 2008). In all of these studies, CODD is described as an initial ulcerative lesion at the coronary band followed by extensive under-running of the horn, with subsequent loss of the hoof capsule. The lesions observed in the seven other goats examined differed by their severity, appearing to have progressed to the sole
of the foot. These were characterised by extensive horn loss on the sole of the foot differing from footrot in that the underlying exposed tissue appearing haemorrhagic and granulomatous. These resembled CODD but were appeared to show a further progressed clinical picture. The strong association between lesion description and incidence and the presence of DD-associated treponemes in the lesions, and their absence in healthy foot tissue, would suggest the involvement of treponemes in the disease process, as considered for DD in dairy cattle, beef cattle and sheep (Evans and others 2008, 2009, Sayers and others 2009, Sullivan and others 2013). The detection rate of the DD-associated treponemes the authors found in the goat lesions is similar to that reported in cattle DD lesions and sheep CODD lesions. Moore and others (2005) identified treponemes in 70 per cent of CODD lesions, and Sayers and others (2009) cultured treponemes from seven of 10 cases of CODD in sheep. In cattle DD samples tested, Evans and others (2009) found that “T. medium/T. vincentii-like”, “T. phagedenis-like” and T. pedis spirochaetes were present in 96.1 per cent, 98 per cent and 76.5 per cent of DD lesions, respectively, with all lesions having at least two BDD treponemes associated. These results are extremely similar to the findings in this study in terms of the percentage detection of
TABLE 1: PCR detection of treponemes in goat foot lesion biopsies (samples 1–10) and healthy goat foot tissue (samples 11–20) Group-specific PCR*
Sample number
Biopsy site (foot)
Type
1
2
3
Treponema genus-specific PCR
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Hind left Front right Hind right Hind right Front left Front left Front left Hind right Hind right Front left Back left Back right Back left Back right Back left Back right Back left Back right Back left Back right
CODD-like lesion CODD-like lesion CODD-like lesion CODD-like lesion CODD-like lesion CODD-like lesion CODD-like lesion CODD-like lesion Severe CODD-like with under-run sole Severe CODD-like with under-run sole Healthy tissue Healthy tissue Healthy tissue Healthy tissue Healthy tissue Healthy tissue Healthy tissue Healthy tissue Healthy tissue Healthy tissue
+ + + + + + + − + + − − − − − − − − − −
+ + + + + + + − + + − − − − − − − − − −
− + + + − + + + + + − − − − − − − − − −
+ + + + + + + + + + + + + + + − + − + −
Treponemes isolated† IF G2JD IF IF IF G6JD G7JD IF G9JD G10JD NIA NIA NIA NIA NIA NIA NIA NIA NIA NIA
*Groups 1, 2 and 3 are Treponema medium/Treponema vincentii-like, Treponema phagedenis-like and Treponema pedis spirochaetes, respectively, which are routinely found in DD lesions. †All isolations are shown for comparison with PCR results. If isolation was successful, the isolated strains are listed. CODD, contagious ovine digital dermatitis; IF, isolation failed; NIA, no isolation attempted.
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FIG 2: A maximum likelihood tree based on 16S rRNA gene sequence comparisons of ∼1,000 aligned bases showing the relationship between the strains isolated here (shown in bold) from goat foot lesions and other DD associated and commensal treponeme 16S rRNA gene sequences. Bootstrap confidence levels are shown as percentages of nodes, and only values above 70% are shown.* = previously reported 16S rRNA gene sequences from BDD lesions; # = previously reported 16S rRNA gene sequences from human oral periodontal infections.
all three Treponema phylogroups with all three Treponema phylogroups were present in 70 per cent of goat lesions, versus 74.5 per cent of cattle DD lesions tested, and 100 per cent of cattle DD lesions were positive for at least one of the DD-associated Treponema phylogroups and as were the goat lesions investigated. The isolation from the goat lesions of three treponemes which are 100 per cent identical to the “T. phagedenis-like” spirochaetes, consistently found within dairy cattle DD lesions (Evans and others 2008, 2009), also suggests a role for these pathogenic bacteria within these lesions. The isolation of two treponemes which are part of the T. pedis spirochaetal group, but show small nucleotide changes, could suggest there are host species-specific variations within this treponeme phylogroup. In this condition, as in CODD in sheep, it is not clear if the treponemes are the primary infection causing the lesion or whether they are secondary infections of an established lesion (of unknown aetiology) which change the nature of the Veterinary Record | March 14, 2015
infection and the clinical outcome. For example, there is some question over the possible interaction of other organisms with CODD such as Dichelobacter nodosus and Fusobacterium necrophorum (Moore and others 2005).To fully understand the aetiology of this foot disease in goats, more studies investigating other bacteria would be necessary. However, the isolation of similar treponemes to those isolated from BDD and CODD lesions and the high detection rate of these DD-associated phylotypes in the lesions would support the hypothesis that this novel foot condition is highly associated with infection by DD treponemes. The prevalence and severity of the disease the authors have reported on just one UK goat dairy farm are very worrying and justify further investigations in future. Further studies are needed to identify effective means of prevention and/ or treatment to reduce spread to other goat herds in the UK and elsewhere. This study suggests that DD may have emerged in yet another host species and vigilance is suggested in goat herds and in other domesticated species.
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Paper Acknowledgements This research was funded from a Biotechnology and Biological Sciences Research Council (BBSRC) Responsive Mode Grant (BB/K009443/1) and an HCC, QMS and EBLEX (a division of the Agriculture and Horticulture Development Board (AHDB)) studentship.
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Digital dermatitis treponemes associated with a severe foot disease in dairy goats L. E. Sullivan, N. J. Evans, S. R. Clegg, S. D. Carter, J. E. Horsfield, D. Grove- White and J. S. Duncan Veterinary Record 2015 176: 283 originally published online November 26, 2014
doi: 10.1136/vr.102858 Updated information and services can be found at: http://veterinaryrecord.bmj.com/content/176/11/283
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Paper Digital dermatitis treponemes associated with a severe foot disease in dairy goats L. E. Sullivan, N. J. Evans, S. R. Clegg, S. D. Carter, J. E. Horsfield, D. Grove- White, J. S. Duncan A UK dairy goat herd was assessed after reports of a severe lameness problem of unknown aetiology. A lameness prevalence estimate was produced and individual clinical examination of 15 randomly selected lame goats was performed. Fifteen animals had foot lesions closely resembling contagious ovine digital dermatitis (CODD) in sheep. Eight of the goats examined presented with typical CODD lesions and seven showed what appeared to be a more severe CODD with under-running of the sole. Ten biopsy samples were obtained from the foot lesions and tested by PCR for the three previously isolated digital dermatitis (DD) Treponema phylogroups and culture of treponemes was attempted. Ninety per cent of the biopsy samples were positive for Treponema medium/Treponema vincentii-like spirochaetes and Treponema phagedenis-like DD spirochaetes and 80per cent were positive for Treponema pedis. Spirochaetes were successfully isolated from 50 per cent of lesion samples. Three isolates were identified as belonging to the T. phagedenis-like spirochaetes and two were identified as T. pedis. The frequent isolation of similar treponemes to those isolated from bovine digital dermatitis and CODD lesions and the identification of these DD-associated phylotypes in the vast majority of lesions support the hypothesis that this novel foot condition is associated with infection by DD treponemes, and given the similarities to CODD, it suggests a causal role. Introduction Lameness in dairy cattle and sheep has serious animal welfare and economic implications (Marshall and others 1991, Enting and others 1997, Hernandez and others 2001, Warnick and others 2001). Lameness is also reported in goats, although with fewer published studies. Prevalence data for lameness in goat herds and diagnosis of causes of lameness have been reported from several countries. In France, Mazurek and others (2007) reported that out of 108 goats, 12.5 per cent were lame and Christodoulopoulos (2009) identified a prevalence of 24 per cent in a herd of 170 goats in Greece. In the UK, Hill and others (1997) found an average lameness prevalence of 9.1 per cent (range 2.7–23.4 per cent) on four goat farms. In a study of 24 dairy goat farms in England and Wales, the mean lameness prevalence was 19.2 per cent (IQR 7.7–30.2 per cent). One of the goat herds visited Veterinary Record (2014) L. E. Sullivan, N. J. Evans, S. R. Clegg, S. D. Carter, Department of Infection Biology, Institute of Infection and Global Health, School of Veterinary Science, University of Liverpool, Science Park ic2, 146 Brownlow Hill, Liverpool, Merseyside L3 5RF, UK J. E. Horsfield, Lanes Veterinary Group, Green Lane Veterinary Centre, Leachfield Industrial Estate, Green Lane West, Cabus,
doi: 10.1136/vr.102858 Garstang PR3 1PR, UK D. Grove- White, J. S. Duncan, Department of Epidemiology and Population Health, Institute of Infection and Global Health, University of Liverpool, Leahurst Campus, Neston, Wirral CH64 7TE, UK E-mail for correspondence: [email protected] Provenance: not commissioned; externally peer reviewed Accepted November 3, 2014
had a lameness prevalence of 52.5 per cent, while three of the herds had no detectable lameness at the time of the visit (Anzuino and others 2010). More recently, similarly high levels of lameness (40 per cent and 67 per cent) were observed among dairy goats on two UK goat farms (Groenevelt and others 2013). Foot disorders recognised in goats are similar to those described in sheep (Winter 2011) and include: interdigital dermatitis, footrot (Duran and others 1990), heel horn erosion (Christodoulopoulos 2009), white line disease, foreign body penetrations (Mgasa and Arnbjerg, 1993) and overgrown feet (Hill and others 1997). In the study by Groenevelt and others (2013), laminitis accompanied by infection with Treponema bacteria was identified as the cause of lameness in two dairy goat herds. Contagious ovine digital dermatitis (CODD) is an emerging severe foot disease of sheep which was first reported in the UK in 1997 (Harwood and others 1997, Davies and others 1999). It has been shown to be associated with Treponema bacteria (Sayers and others 2009) and is hypothesised to have derived from digital dermatitis (DD) lesions in dairy cows (Dhawi and others 2005). CODD is now widespread in UK sheep (Kaler and Green 2008, Angell and others 2014). Although goats and sheep share many foot diseases, CODD is yet to be reported in goats; however, having possibly crossed into sheep from one host species, there was always a chance that this could happen again and infection of other domesticated species could occur. Here, the authors report the clinical and microbiological findings of a severe lameness problem in a UK dairy goat herd. Clinical descriptions of lesions were produced and lesion biopsies were investigated by PCR and bacterial culture for an association with the same Treponema bacteria found in bovine digital dermatitis (BDD) and CODD. March 14, 2015 | Veterinary Record
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Paper Materials and methods Farm details and clinical description of lesions Following reports of a severe lameness problem of unknown aetiology, a UK goat herd was assessed by the attending veterinary surgeons ( JSD and JEH) in October 2013. The herd consisted of 1000 milking goats of Anglo-Nubian and British Saanen breed. The goats were housed year round in four straw yards (250 goats per yard), which were cleaned out every six to eight weeks. Milking occurred twice a day and goats were fed a ration of grass silage and cereal-based concentrate. Infection with caprine arthritis encephalitis virus had previously been diagnosed on the farm. All goats were routinely foot trimmed every three months and the herd walked through a copper sulphate footbath post milking, twice weekly. The farmer estimated that 30 per cent of the herd were lame and his current treatment for lame goats was foot trimming and topical application of oxytetracycline spray; in some but not all lameness cases the goats would also be treated with a course of the parental antibiotic, ceftiofur (Naxcel, Zoetis). However, it appeared this was not controlling the disease effectively and a high level of lameness was apparent on the farm for several months. The prevalence of lameness in this herd was estimated by the attending veterinary surgeon after lameness scoring the entire herd on exit from the milking parlour. Due to the rate and numbers of goats exiting the parlour, a simple lame/not lame scoring system was used (Phythian and others 2013). On this basis, 65 per cent of the herd were identified as lame. There appeared to be no observable difference in lameness prevalence between the four straw yards. Fifteen lame goats were randomly selected for further examination. All were non-weight bearing and were affected on one leg only. Eight of the 15 lame goats had foot lesions showing very close resemblance to the typical presentation of CODD in sheep. These goats displayed separation of the hoof capsule at the level of the coronary band with the underlying exposed tissue appearing haemorrhagic and granulomatous (Fig 1a, b). These lesions appeared to originate at the coronary band. The lesions on the other seven goats examined demonstrated an even more severe presentation, with loss of solar horn accompanied by marked granulation and haemorrhage of the sole (Fig 1c). The lesions on these seven goats therefore presented lesions similar to CODD, but the origin of the lesions was indistinguishable due to their severity.
Sample collection Ten of the goats with foot lesions were chosen randomly from the goats examined and the lesions were biopsied using a 3 mm punch biopsy under local anaesthesia (Evans and others 2008). Lesion samples were divided into two with half transferred into transport medium and placed on ice for subsequent Treponema culture. Transport medium consisted of oral treponeme enrichment broth (OTEB; Anaerobe Systems, Morgan Hill, California, USA) and the antibiotics rifampicin (5 μg/ml) and enrofloxacin (5 μg/ml). The remaining tissue from lesions (for PCR analysis) was transported on ice and stored at −20° C. Healthy goat foot tissues (n=10) were used to serve as negative controls. Single rear-hoof skin biopsies were collected from each hindfoot of five dairy goats sent to slaughter that did not have any evidence of foot lesions. All healthy tissue samples were transported and stored as above.
DNA extraction and treponeme PCR assays For PCR analysis, frozen tissues from the lesions and healthy tissues were thawed and DNA extracted using a DNeasy Kit (Qiagen, UK) according to the manufacturer’s instructions, and genomic DNA was stored at −20° C. Samples were subjected to nested PCR assays specific for the three DD-associated treponeme groups, “Treponema medium/Treponema vincentii-like”, “Treponema phagedenis-like” and Treponema pedis described by Evans and others (2008, 2009) with resulting PCR products Veterinary Record | March 14, 2015
encompassing 300–500 bp of the 16S rRNA gene. All foot samples were also subjected to the Treponema genus PCR assay (Moore and others 2005).
Culture of spirochaetes Bacterial isolation was attempted on all lesion samples (n=10). A (1–1.5 mm) piece of tissue was transferred into an anaerobic cabinet (85 per cent N2, 10 per cent H2 and 5 per cent CO2, 36° C). Each was inoculated into OTEB (Anaerobe Systems) with 10 per cent fetal calf serum (FCS) and the antibiotics rifampicin (5 μg/ml) and enrofloxacin (5 μg/ml). After two to five days, bacteria were then subcultured on fastidious anaerobe agar plates (LabM, Bury, UK) with 5 per cent defibrinated sheep blood, 10 per cent FCS and antibiotics as above. After one to two weeks, single colonies were inoculated into growth media and were checked for pure culture by phase contrast microscopy. DNA was extracted from treponeme cultures and the isolated organisms were identified using a 16S rRNA gene PCR and gene sequencing as previously described (Evans and others 2008). Bacterial isolation was not attempted on healthy tissues following treponeme PCR assay results.
Phylogenetic analysis of spirochaete isolates To understand the relationship of the isolated spirochaetes with other treponemes, a phylogenetic tree was produced from the aligned and trimmed 16S rRNA gene PCR product sequences from the isolates and relevant microorganisms are available in GenBank. Sequences were assembled into a double-stranded consensus sequence using Chromas Pro 1.41 (Technelysium Pty Ltd). Consensus sequences were aligned by Clustal W and trimmed using Mega 5.2 (Tamura and others 2011). For tree analysis, the most appropriate evolution model was predicted using ‘model test’ as implemented in the TOPALi program(Milne and others 2009). The final model for nucleotide substitutions chosen was the general time reversal model (Tavare 1986), used to infer a bootstrapped maximum likelihood tree; bootstrapping was performed 10, 000 times.
Results Treponeme PCR assays The 10 biopsy samples obtained from the foot lesions were all positive for the Treponema genus-specific PCR (Table 1, samples 1–10), which detects all Treponema species, including pathogens and commensals. The DD group-specific PCR assays found T. medium/T. vincentii-like spirochaetes and T. phagedenis-like DD spirochaetes in 90 per cent of samples and 80 per cent were positive for T. pedis. All lesional samples were positive for at least one and usually more of the DD-associated Treponema phylogroups upon PCR analysis (Table 1). All healthy foot tissues were negative for all three of the DD group-specific PCR assays. Of the 10 healthy foot tissue samples, seven were positive for the Treponema genus-specific PCR.
Isolation of spirochaetes Spirochaetes were successfully isolated from 50 per cent of cultured lesion samples (n=10) (Table 1). The isolates G6JD, G7JD and G10JD (GenBank accession codes: KJ206529, KJ206530, KJ206532, respectively) were identified as belonging to the “T. phagedenis-like” spirochaetes and shared 100 per cent 16S rRNA gene sequence identity with the “T. phagedenis-like” DD spirochaete strain T320A (GenBank accession: EF061261), previously isolated from a dairy cow DD lesion in the UK (Evans and others 2008). Isolates G2JD and G9JD (GenBank accession codes: KJ206528, KJ206531, respectively) belonged to the T. pedis spirochaetes and shared 99 per cent 16S rRNA gene sequence identity with T. pedis T3552B (NR 044064) previously isolated from a dairy cow DD lesion in the UK (Evans and others 2008).
Phylogenetic analysis of isolated spirochaetes All successfully cultured isolates were clustered with their respective closest spirochaete relatives upon phylogenetic
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Paper
FIG 1: (a) “CODD type lesion” in a dairy goat. (b) “CODD type lesion” in a goat with separated hoof horn removed to show underlying granulomatous and haemorrhagic tissue. (c) Goat foot with a more severe “CODD type lesion” affecting sole of foot.
analysis (Fig 2). Goat isolates from samples 6, 7 and 10 were clustered closely with the “T. phagedenis-like” spirochaetes, as would be expected from the PCR analyses of the lesional material. Samples 2 and 9 were clustered with the T. pedis spirochaetes; however, they formed a separate clade likely due to five nucleotide substitutions in the 16S rRNA gene (A69G, T73C, A219G, T405C, A440G).
Discussion The foot disease documented here was extremely severe with no early lesions identified; although in the majority of cases it appeared that the lesions originated at the coronary band, as with CODD in sheep (Davies and others 1999, Winter 2008). The lesions in eight of the goats examined (Fig 1a, b), where there was separation of hoof horn at the level of the coronary band accompanied by extensive under-running of the hoof horn capsule, bear a marked similarity to the published lesion descriptions of CODD in sheep (Davies and others 1999, Winter 2008). In all of these studies, CODD is described as an initial ulcerative lesion at the coronary band followed by extensive under-running of the horn, with subsequent loss of the hoof capsule. The lesions observed in the seven other goats examined differed by their severity, appearing to have progressed to the sole
of the foot. These were characterised by extensive horn loss on the sole of the foot differing from footrot in that the underlying exposed tissue appearing haemorrhagic and granulomatous. These resembled CODD but were appeared to show a further progressed clinical picture. The strong association between lesion description and incidence and the presence of DD-associated treponemes in the lesions, and their absence in healthy foot tissue, would suggest the involvement of treponemes in the disease process, as considered for DD in dairy cattle, beef cattle and sheep (Evans and others 2008, 2009, Sayers and others 2009, Sullivan and others 2013). The detection rate of the DD-associated treponemes the authors found in the goat lesions is similar to that reported in cattle DD lesions and sheep CODD lesions. Moore and others (2005) identified treponemes in 70 per cent of CODD lesions, and Sayers and others (2009) cultured treponemes from seven of 10 cases of CODD in sheep. In cattle DD samples tested, Evans and others (2009) found that “T. medium/T. vincentii-like”, “T. phagedenis-like” and T. pedis spirochaetes were present in 96.1 per cent, 98 per cent and 76.5 per cent of DD lesions, respectively, with all lesions having at least two BDD treponemes associated. These results are extremely similar to the findings in this study in terms of the percentage detection of
TABLE 1: PCR detection of treponemes in goat foot lesion biopsies (samples 1–10) and healthy goat foot tissue (samples 11–20) Group-specific PCR*
Sample number
Biopsy site (foot)
Type
1
2
3
Treponema genus-specific PCR
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Hind left Front right Hind right Hind right Front left Front left Front left Hind right Hind right Front left Back left Back right Back left Back right Back left Back right Back left Back right Back left Back right
CODD-like lesion CODD-like lesion CODD-like lesion CODD-like lesion CODD-like lesion CODD-like lesion CODD-like lesion CODD-like lesion Severe CODD-like with under-run sole Severe CODD-like with under-run sole Healthy tissue Healthy tissue Healthy tissue Healthy tissue Healthy tissue Healthy tissue Healthy tissue Healthy tissue Healthy tissue Healthy tissue
+ + + + + + + − + + − − − − − − − − − −
+ + + + + + + − + + − − − − − − − − − −
− + + + − + + + + + − − − − − − − − − −
+ + + + + + + + + + + + + + + − + − + −
Treponemes isolated† IF G2JD IF IF IF G6JD G7JD IF G9JD G10JD NIA NIA NIA NIA NIA NIA NIA NIA NIA NIA
*Groups 1, 2 and 3 are Treponema medium/Treponema vincentii-like, Treponema phagedenis-like and Treponema pedis spirochaetes, respectively, which are routinely found in DD lesions. †All isolations are shown for comparison with PCR results. If isolation was successful, the isolated strains are listed. CODD, contagious ovine digital dermatitis; IF, isolation failed; NIA, no isolation attempted.
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Paper
FIG 2: A maximum likelihood tree based on 16S rRNA gene sequence comparisons of ∼1,000 aligned bases showing the relationship between the strains isolated here (shown in bold) from goat foot lesions and other DD associated and commensal treponeme 16S rRNA gene sequences. Bootstrap confidence levels are shown as percentages of nodes, and only values above 70% are shown.* = previously reported 16S rRNA gene sequences from BDD lesions; # = previously reported 16S rRNA gene sequences from human oral periodontal infections.
all three Treponema phylogroups with all three Treponema phylogroups were present in 70 per cent of goat lesions, versus 74.5 per cent of cattle DD lesions tested, and 100 per cent of cattle DD lesions were positive for at least one of the DD-associated Treponema phylogroups and as were the goat lesions investigated. The isolation from the goat lesions of three treponemes which are 100 per cent identical to the “T. phagedenis-like” spirochaetes, consistently found within dairy cattle DD lesions (Evans and others 2008, 2009), also suggests a role for these pathogenic bacteria within these lesions. The isolation of two treponemes which are part of the T. pedis spirochaetal group, but show small nucleotide changes, could suggest there are host species-specific variations within this treponeme phylogroup. In this condition, as in CODD in sheep, it is not clear if the treponemes are the primary infection causing the lesion or whether they are secondary infections of an established lesion (of unknown aetiology) which change the nature of the Veterinary Record | March 14, 2015
infection and the clinical outcome. For example, there is some question over the possible interaction of other organisms with CODD such as Dichelobacter nodosus and Fusobacterium necrophorum (Moore and others 2005).To fully understand the aetiology of this foot disease in goats, more studies investigating other bacteria would be necessary. However, the isolation of similar treponemes to those isolated from BDD and CODD lesions and the high detection rate of these DD-associated phylotypes in the lesions would support the hypothesis that this novel foot condition is highly associated with infection by DD treponemes. The prevalence and severity of the disease the authors have reported on just one UK goat dairy farm are very worrying and justify further investigations in future. Further studies are needed to identify effective means of prevention and/ or treatment to reduce spread to other goat herds in the UK and elsewhere. This study suggests that DD may have emerged in yet another host species and vigilance is suggested in goat herds and in other domesticated species.
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Paper Acknowledgements This research was funded from a Biotechnology and Biological Sciences Research Council (BBSRC) Responsive Mode Grant (BB/K009443/1) and an HCC, QMS and EBLEX (a division of the Agriculture and Horticulture Development Board (AHDB)) studentship.
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Digital dermatitis treponemes associated with a severe foot disease in dairy goats L. E. Sullivan, N. J. Evans, S. R. Clegg, S. D. Carter, J. E. Horsfield, D. Grove- White and J. S. Duncan Veterinary Record 2015 176: 283 originally published online November 26, 2014
doi: 10.1136/vr.102858 Updated information and services can be found at: http://veterinaryrecord.bmj.com/content/176/11/283
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