Isolation and Characterisation of a Novel Spirochaete from Severe Virulent Ovine Foot Rot

J. Med. Microbiol. Ð Vol. 50 2001), 1061±1068 # 2001The Pathological Society of Great Britain and Ireland ISSN 0022-2615 BACTERIAL PATHOGENICITY Isolation and characterisation of a novel spirochaete from severe virulent ovine foot rot I. DEMIRKAN, S. D. CARTER, C. WINSTANLEYÃ,K.D.BRUCE{,N.M.McNAIR{,M.WOODSIDE} andC.A.HARTÃ Departments of Veterinary Immunology and ÃMedical Microbiology and Genitourinary Medicine, University of Liverpool, Liverpool, {Division of Life Sciences, Franklin-Wilkins Building, Kings College, London, {Veterinary Research Laboratory, Belfast, Northern Ireland and }Clare Veterinary Group, Ballyclare, Northern Ireland A novel spirochaete was isolated from a case of severe virulent ovine foot rot SVOFR) by immunomagnetic separation with beads coated with polyclonal anti-treponemal antisera and prolonged anaerobic broth culture. The as yet unnamed treponeme differs considerably from the only other spirochaete isolated from ovine foot rot as regards morphology, enzymic pro®le and 16S rDNA sequence. On the basis of 16S rDNA, it was most closely related to another unnamed spirochaete isolated from cases of bovine digital dermatitis in the USA, raising the possibility of cross-species transmission. Further information is required to establish this novel ovine spirochaete as the cause of SVOFR. Introduction of cattle, bovine digital dermatitis, in the USA [11], Germany [12] and the UK [13]. The latter study used Severe virulent ovine foot rot SVOFR) is a recently immunomagnetic separation to isolate a spirochaete identi®ed and important disease of sheep, and an from affected cattle [13]. The present study employed a increasing cause of lameness in sheep in the UK [1]. similar approach in a case of SVOFR and described the SVOFR is a chronic, necrotising disease of the epi- morphological, biochemical and genotypic character- dermis of the interdigital skin, in some cases involving istics of the novel treponeme isolated. the entire hoof matrix. As severely affected tissue is destroyed the hooves become detached from the underlying matrix of the foot, resulting in lameness [2]. Materials and methods Various aetiological agents of ovine foot rot have been A ¯ock of sheep from Northern Ireland with epidemic described and it is thought that initial invasion of the lameness due to SVOFR was selected for study. This epidermis by Dichelobacter nodosus and Fusobacter- ¯ock had no known prior contact, directly or indirectly, ium necrophorum subsp. necrophorum paves the way with cattle. A biopsy c.1g) of a lesion was taken from for invasion by secondary opportunists including the right rear foot and washed in sterile phosphate- Bacteroides fragilis, Prevotella spp. and several other buffered saline PBS), pH 7.4. Care was taken to strict anaerobes [3±7]. Spirochaetes have been ob- ensure that the biopsy was full skin thickness, served over many years in cases of foot rot in pigs, including both epidermis and dermis. After washing, horses and sheep [3, 8]. In a recent microbiological the biopsy was immediately placed in Oral Treponema investigation of unusually severe ovine foot rot [1], D. Enrichment Broth OTEB; Anaerobe Systems, Morgan nodosus was not detected but F. necrophorum, Bact. Hill, CA, USA) supplemented with rifampicin 5 mg=L fragilis and Prevotella spp. were isolated [9]. Motile and nalidixic acid 500 mg=L. The biopsy in OTEB was spirochaetes were also observed and successfully shipped to Liverpool by surface mail 48-h journey cultured [9, 10]. Recently, spirochaetes have been time). On arrival, the biopsy was placed in an cultured successfully from cases of a related disease anaerobic cabinet maintained at 378C and diced into small fragments with sterile instruments. To check Received 2 April 2001; accepted 5 June 2001. viability, a smear of the material was examined by Corresponding author: Professor C. A. Hart e-mail: dark-®eld microscopy and was found to contain motile [email protected]). spirochaetes. The rest of the material was placed in 1062 I. DEMIRKAN ET AL. fresh antibiotic-containing OTEB supplemented with H2O2 3% v/v and observing it for evolution of bubbles fetal calf serum FCS) 10% v/v and incubated in an of oxygen. anaerobic cabinet Don Whitley Scienti®c, Shipley, West Yorkshire) at 378C for 24 h. DNA analysis DNA was extracted from 7-day OTEB antibiotic-free) Immunomagnetic separation cultures 20 ml). Bacteria were pelleted by centrifuga- The method used was as described previously for tion at 12 000 g for 20 min and washed twice in PBS. the isolation of a spirochaete from a case of bovine The pellet was suspended in 1ml of lysis buffer digital dermatitis [13]. Brie¯y, immunomagnetic beads 500 mM Tris, pH 9; 20 mM EDTA; 10 mM NaCl; 2.8 ìm diameter) covalently coated with polyclonal sodium dodecyl sulphate 1% w/v and freshly prepared anti-rabbit IgG were obtained from Dynech Billings- proteinase K 0:5mg=ml) and incubated at 428C for 4 h hurst, Sussex). The beads 1:5 3 107=ml) were incu- on a shaker. DNA was extracted with phenol, bated overnight at 48C with a mixture of polyclonal precipitated with 5 M NaCl and absolute ethanol, rabbit antiserum to Treponema denticola and T. vincentii suspended in 100 ìl distilled water and stored at 200 ìl of each). After washing in PBS, the coated À208C. beads 200 ìl) were mixed with the 24-h biopsy culture 1ml) by slow tilting and rotation for 60 min at 4 8C. 16S rRNA gene sequencing and analysis The beads were then collected by means of a magnetic particle concentrator and the supernate was discarded. Primers pA pA: 59-AGA GTT TGA TCC TGG CTC The beads were washed in PBS incorporating FCS 10% AG-39) and pH9 pH9:59-AAG GAG GTG ATC CAG v/v and re-collected magnetically. The beads were then CCG CA-39), developed by Edwards et al. [14] and placed into fresh OTEB supplemented with antibiotics supplied by MWG-BIOTECH UK Milton Keynes) as above and some were streaked on to Fastidious were used to amplify 16S rDNA as follows. Genomic Anaerobe Agar incorporating de®brinated sheep blood DNA 1 ìl) was used directly in 50-ìl volumes 5% v/v FABA) plates LabM, Bury), supplemented containing Taq DNA polymerase Roche, Lewes, East with rifampicin 1mg/L and enro¯oxacin 1mg/L. Broth Sussex) 1.25 U, 200 nM of each primer pA and pH9), and plates were incubated in an anaerobic cabinet at 13 PCR reaction buffer supplied by Roche) and 378C for 7 days. After initial isolation the culture was 100 ìM nucleotides dATP, dCTP, dGTP, dTTP). continued in OTEB without added antimicrobial agents. Ampli®cations were performed in a Perkin Elmer The isolated treponomes were stored on Protect Beads 2400 thermal cycler Applied Biosystems, Warrington, Technical Service Consultants, Heywood) at À808C Cheshire) for 30 cycles consisting of 958C 1min), until required for further analysis. 558C 1min) and 72 8C 2 min) with an additional extension time at 728C 10 min) after completion of 30 cycles. At the end of the ampli®cation, 8-ìl samples Electron microscopy were subjected to electrophoresis on a standard TAE agarose 0.7% w/v gel and stained after electrophoresis Carbon re-inforced, formvar-coated copper specimen with 2 ìl of ethidium bromide 10 mg=ml in 200 ml of support grids 400 square mesh) were coated with a TAE buffer, to con®rm the presence of an ampli®ed drop of a 48-h OTEB ovine spirochaete culture product. This sample was puri®ed with QIAquick PCR suspension. The grid was air-dried then washed three puri®cation columns Qiagen, Crawley, West Sussex) times in a drop of distilled water on a microscope slide and subjected to sequencing at King's College Oral to remove crystallised salts). It was then negatively Microbiology Department with oligonucleotide primers stained with EM grade potassium phosphotungstate, pH pA and pH9. The accession code number for this 7.0 Agar Scienti®c, Standstead) 1%. Grids were ex- sequence is AF363634. amined with a Philips 301electron microscope. The phylogenetic relationship of the ovine sequence Enzyme pro®le was established by comparison with 28 treponeme and related sequences, taken from the Ribosomal Database A 48-h OTEB culture 10 ml) without antibiotics was Project RDP) [15], selected to cover this group within centrifuged at 12 000 g for 20 min. The supernate was the Bacterial domain. In addition to these sequences, removed and the pellet was washed twice in PBS, pH the two sequences providing the closest currently 7.2. The pellet was suspended in PBS to a density available FASTA matches Wisconsin package version equivalent to MacFarland turbidity standard 5. The 10.1, Genetics Computer Group GCG), Madison, WI, suspension was inoculated into cupules of the API- USA) to the ovine sequence were also included, along ZYM strip system bioMeÂrieux, Basingstoke), which with two sequences from treponemes identi®ed pre- contains 19 different substrates. The strip was incu- viously as causing similar disease [10, 12]. Following bated and reactions were interpreted according to the alignment of these sequences, a dendrogram was manufacturer's instructions. Catalase activity was generated from the 1326 aligned bases by dnaml determined by mixing a portion of the pellet with PHYLIP version 3.573c ± updated from Felsenstein NOVEL SPIROCHAETE FROM OVINE FOOT ROT 1063 [16]). The information from this dnaml analysis is 3 mm diameter) and with irregular ®lamentous edges displayed as a phylogram generated by Tree View after longer incubation. A zone of â-haemolysis was version 1.6.1) software [17], with Escherichia coli 16S visible around the colonies. On dark-ground micro- rDNA sequence data providing information for the scopy the bacteria were highly motile, exhibiting outgroup. corkscrew-like and ¯exuous motility. The spirochaete has been designated G179.

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