The 16S rRNA genes from spirochaetes associated with digital dermatitis of British cattle were amplified by polymerase chain reaction from digital dermatitis lesion biopsies using one universal and one treponeme-specific primer. Two treponemal sequences were identified both of which shared a high degree of homology with the oral pathogen Treponema denticola (98%). Two further 16S rRNA gene sequences were obtained and shared similarity to Bacteroides levii (99%) and Mycoplasma hyopharyngis (98%). Polymerase chain reaction with T. denticola-specific primers amplified a potential virulence gene from digital dermatitis lesions which shared a high degree of homology to the 46-kDa haemolysin gene of T. denticola. The significance of the presence of organisms in digital dermatitis lesions of the bovine foot which are closely related to oral pathogens is discussed.
Digital dermatitis (DD) is an infectious disease of cattle characterised by an ulcerative lesion of the bovine foot that is often associated with lameness . The disease was first described in Italian cattle in 1974  and has been recognised in Great Britain since 1988 . The aetiology of DD is unknown but several studies have implicated spirochaetes. An unknown Treponema species has been identified in DD lesions by electron microscopy and the flagellum of this spirochaete shared an epitope with the flagellum of the Lyme disease spirochaete, Borrelia burgdorferi. Furthermore, antibodies from dairy cows infected with DD were reactive with B. burgdorferi whole cell antigens . Recently spirochaetes have been isolated from American dairy cows with DD and analysis of the enzymatic activity showed that these isolates belonged to the genus Treponema. The phenotypes of two distinct isolates were characterised by colony morphology and protein electrophoresis patterns, but the taxonomic position of these isolates remains unclear. Choi et al.  identified five treponemal phylotypes which shared identity with Treponema phagedenis, T. denticola and T. vincentii by using sequence analysis of 16S rRNA genes PCR amplified from biopsy material of DD lesions. Additionally, these authors showed that T. denticola-like spirochaetes were most commonly associated with DD lesions by in situ hybridisation with fluorescently labelled oligonucleotide probes. In this laboratory, we demonstrated the apparent presence of two closely related treponemal phylotypes which shared close homology to T. denticola based on 16S rRNA gene sequence analysis of PCR-amplified DNA.
T. denticola is considered a major member of the pathogenic periodontal microbiota and several virulence genes of T. denticola have been described including those encoding proteases . A 46-kDa haemolysin gene has been characterised from T. denticola and the protein has been shown to produce haemolysis and haemoxidation of sheep erythrocytes . Thus the aim of this work was to classify spirochaetes from DD lesions of British dairy cows and to determine whether they possessed a homologue of the 46-kDa haemolysin gene of T. denticola.
Materials and methods
Preparation of samples for PCR
Post-mortem specimens of epidermis were obtained from characteristic DD lesions in two British dairy cows. Material for PCR was frozen and stored at −20°C until required. Biopsies were cut into cubes approximately 5 mm in length and three cubes were incubated in 3 ml of lysis buffer (500 mM Tris pH 9; 20 mM EDTA; 1% SDS and 0.5 mg ml−1 proteinase K) at 60°C for 24 h in order to release total DNA. Cellular debris was removed by centrifugation at 2000×g for 10 min and the supernatant was extracted twice with an equal volume of buffered phenol (Gibco BRL, Life Technologies). DNA was precipitated from the aqueous layer by the addition of 0.1 volumes of 3 M sodium acetate pH 8.0 and 2 volumes of ethanol. The DNA was recovered by centrifugation at 13 000×g for 30 min at 4°C and washed with 70% ethanol. The pellet was air dried, dissolved in water, and stored at −20°C .
Polymerase chain reaction
Primers were designed to amplify approximately 1500 bp of the treponemal 16S rRNA gene. The forward and reverse primer sequences are given in Table 1. PCR reactions were performed in the following manner. A 50 μl reaction mixture was set up containing Thermo DNA polymerase reaction buffer (Promega), 200 μM dNTPs (Promega), 1.5 mM MgCl2, 2.5 units of Taq DNA polymerase (Promega), 10 pmol of each primer and 2 μl of the processed sample. The mixture was overlaid with 50 μl of light mineral oil. Cycling was carried out in a thermal cycler (Biometra) with the following touchdown cycle: an initial denaturation step of 3 min at 94°C, followed by two cycles of 1 min at 94°C, 1 min at 64°C, 2.5 min at 72°C. These cycles were repeated but with the annealing temperature reduced by 2°C every two cycles until the annealing temperature was 54°C. Then 35 cycles of 1 min at 94°C, 1 min at 54°C, 2.5 min at 72°C were performed followed by a final incubation of 72°C for 5 min. Negative controls were included with each set of samples. The completed PCR reactions were analysed by agarose gel electrophoresis on a 1% agarose gel in 40 mM Tris-acetate/1 mM EDTA buffer (TAE).
|16S rRNA forward||5′-AGAGTTTGATCMTGGCTCAG|
|16S rRNA reverse||5′-ACGGCTACCTTGTTACGACTTCAC|
|16S rRNA forward||5′-AGAGTTTGATCMTGGCTCAG|
|16S rRNA reverse||5′-ACGGCTACCTTGTTACGACTTCAC|
Cloning of PCR products
PCR products were purified from agarose gel slices containing the desired band using Wizard PCR Preps purification kit (Promega). The purified PCR product was made blunt-ended using Pfu polymerase to infill any overhangs and the resultant DNA was ligated with linearised pCR-Script vector (Stratagene Inc.) using the manufacturer's protocols. The ligated mixture was used to transform MaxEfficiency competent Escherichia coli cells (Gibco BRL). Recombinant plasmids were selected by blue-white screening after plating onto LB medium containing ampicillin (100 μg ml−1) containing X-gal and IPTG. Plasmid minipreps of selected recombinants were restriction digested to ensure that they contained inserts of the correct size.
Nucleotide sequencing and analysis
Recombinant pCR-Script clones were prepared for fluorescent cycle sequencing using Wizard Minipreps (Promega) with an additional isopropanol precipitation step for increased purity. Cycle sequencing was performed using the Taq Terminator FS kit (Perkin-Elmer) in the following manner. In a volume of 20 μl were added 450 ng of the purified plasmid, 3.2 pmol of sequencing primer and 8 μl of Taq Terminator Ready Reaction Mix. The mixture was overlaid with 50 μl of mineral oil and subjected to the following program in a thermal cycler (Perkin-Elmer): 30 cycles of 15 s at 95°C, 30 s at 50°C and 3 min at 60°C. Unincorporated dye terminators were removed from the completed sequencing reactions by precipitation with ethanol. The pellets were dissolved in gel loading buffer and analysed on an ABI Model 373A automated DNA sequencer (Applied Biosystems Inc.). Trace data were analysed and assembled into contiguous sequences using DNAStar software (DNAStar Inc.).
Phylogenetic analysis of 16S rRNA gene sequences
PCR was used to amplify treponemal 16S rRNA gene sequences from biopsy material obtained from the feet of cattle showing the characteristic DD lesions and also from the feet of a healthy animal. Amplified products of the correct size were obtained only from the diseased animal. These products were gel purified and cloned into the pCR-Script vector for further analysis. Fifty clones that contained 1.5 kb 16S rDNA inserts were identified by restriction analysis of purified plasmid DNA. Of these clones, a randomly selected number were subjected to nucleotide sequencing to allow phylogenetic analysis of their 16S rDNA insert sequences. Thus, 20 plasmids containing cloned PCR products were sequenced completely and the resulting 16S rRNA gene sequences were analysed using homology searches of the GenBank database. Fourteen insert sequences were very closely related to T. denticola (98% identity) and these could be subdivided into two groups of identical sequences (DD1 and DD2, eight and six sequences respectively). DD1 and DD2 shared 99% identity to each other. Five insert sequences were identical to each other and shared 99% identity to Bacteroides levii. The one remaining insert sequence had 98% identity to Mycoplasma hyopharyngis. A phylogenetic tree showing the relatedness of the two treponemal sequences DD1 and DD2 to other treponemal phylotypes is shown in Fig. 1.
Nucleotide sequence of the amplified treponemal haemolysin gene
A PCR product of 1.4 kb was amplified from bovine DD lesion material using PCR primers designed from the sequence of the T. denticola 46 kDa haemolysin gene. The same PCR failed to amplify a product of the expected size from the foot of a healthy animal. The product was purified and cloned into the pCR-Script vector for nucleotide sequencing. Nucleotide sequence analysis of a randomly chosen selection of 12 recombinant clones showed that all had identical inserts. This nucleotide sequence shared 89% sequence identity with the T. denticola 46 kDa haemolysin gene (hly) and possessed an open reading frame which encoded a protein of 48 kDa which shared 92% identity with Hly (Fig. 2).
The identification of two treponemes closely related to T. denticola is consistent with the observation of two morphologically distinct groups of spirochaetes in DD lesions by electron microscopy  and the initial findings observed in this laboratory . That several spirochaetes are associated with DD confirms the findings of Choi et al.  who identified five treponemal phylotypes using a similar PCR approach to amplify and sequence 16S rRNA genes. However, we did not find treponemes that were related to any other phylotypes. This may be a result of differences in the age of the biopsied lesions with older lesions possibly being subject to a more widespread secondary infection. The fact that Choi et al. also showed that T. denticola-like treponemes were most commonly found in DD lesions by in situ hybridisation with specific oligonucleotide probes suggests that this phylotype may be the common factor in the pathogenesis of DD. T. denticola is a putative human pathogen associated with periodontal disease and produces a number of virulence factors including a 46 kDa haemolysin. Primers designed on the T. denticola hly gene sequence were used to amplify successfully the expected sized product from a DD lesion biopsy. The nucleotide sequence of this product showed an open reading frame of identical size to that of T. denticola and the deduced peptide sequence had 92% homology with T. denticola Hly. It is significant that the T. denticola-like phylotypes associated with DD lesions possess this virulence gene, whether highly conserved in all or limited in distribution to one or a few, and adds more evidence to the proposed involvement of these treponemes in the aetiology of DD. The identification of organisms closely related to B. levii and M. hyopharyngis, both found orally, in biopsy samples suggests that the bovine foot may mimic the periodontal microenvironment. The possibility that these organisms may have been introduced into the DD lesions from bovine saliva as either primary or secondary colonisers cannot be ruled out.
Until the treponemes associated with digital dermatitis have been cultured, if possible, and infectivity studies have been performed, molecular techniques such as those described here provide the only clues to identifying and characterising the infectious agent(s) responsible for causing DD.