This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Page 1 of 27A c c e p t e d M a n u s c r i p t A previously isolated contagious ovine digital dermatitis spirochete was located within 34 one of the three phylogroups, group 3, and could also be identified within this group on 35 the basis of phenotype testing, suggesting BDD and contagious ovine digital dermatitis 36 may share the same aetiological agent. A strain isolated from a bovine interdigital 37 dermatitis lesion, could be identified as part of BDD isolate group 2, suggesting bovine 38 interdigital dermatitis and BDD may have the same causative agent. Two common 39 enzyme activities, C4 esterase and C8 esterase lipase, were identified in all BDD 40 associated treponemes suggesting common metabolic pathways for sharing this novel 41 niche or even common virulence traits. Further studies are required to determine whether 42 the three groups of novel treponemes are representative of new treponeme taxa and to 43 delineate how they interact with bovine tissues to cause disease. 44

This study used a PCR-based approach targeting 16S rRNA gene fragments to determine the occurrence and association of the three bovine digital dermatitis (BDD) treponeme phylogroups within lesions found in cattle from the United Kingdom. Examination of 51 BDD lesions collected from infected cattle across the United Kingdom revealed that BDD treponeme group 1 (Treponema medium/Treponema vincentii-like), group 2 (Treponema phagedenis-like), and group 3 (Treponema putidum/Treponema denticola-like) were present in 96.1%, 98%, and 76.5% of BDD lesions, respectively. The three phylogroups were present together in 74.5% of lesions. The PCR assays enabled the isolation of further treponeme strains from previously mixed primary BDD lesion cultures. Here a representative from each of the three distinct treponeme phylogroups was isolated from a single BDD lesion for the first time. These data highlight the extent to which this disease is polytreponemal. Immunohistochemistry and electron microscopy were used to investigate lesional hoof tissues, resulting in treponemes being identified copiously in hair follicles and sebaceous glands, suggesting a potential route of exit and/or entry for these pathogens. This study gives further evidence for the importance of the three treponeme groups in BDD pathogenesis and reiterates the value of molecular genetic approaches for isolating and identifying fastidious anaerobes.

Bovine digital dermatitis (BDD) is a debilitating infection that is being increasingly recognized in livestock worldwide. Several treponeme phylotypes have been identified in BDD lesions, although only a single BDD-associated treponeme taxon (Treponema brennaborense) has been proposed thus far. In a previous study, we observed that four BDD-associated spirochaete isolates formed a distinct phylogenetic cluster on the basis of 16S rRNA gene sequence analysis and shared less than 97 % sequence similarity with recognized treponeme species. Further characterization of these isolates on the basis of growth characteristics, flaB2 gene sequences, enzyme profiles and cell morphology confirmed that they formed a coherent taxonomic group displaying marked genotypic and phenotypic differences with respect to recognized treponeme species. The four novel isolates displayed a novel 3 : 6 : 3 flagellar pattern rather than the 2 : 4 : 2 pattern shown by their closest relatives and exhibited esterase C4, esterase lipase C8, trypsin and chymotrypsin enzyme activities. Therefore these four new isolates represent a novel species of the genus Treponema, for which the name Treponema pedis sp. nov. is proposed. The type strain is T3552B T (5DSM 18691 T 5NCTC 13403 T ).Treponemes are typically found in the oral cavity, digestive tract and genital areas of humans and animals (Smibert, 1984). Several treponeme taxa are associated with disease and there is an unequivocal aetiology for certain treponeme infections such as human syphilis and yaws, which are caused by Treponema pallidum subsp. pallidum and T. pallidum subsp. pertenue, respectively (Radolf et al., 2006). In contrast, other treponeme infections, such as human periodontal infections and bovine digital dermatitis (BDD), appear to have a more complicated aetiology involving co-infection by several different treponemes (Choi et al., 1997;Dewhirst et al., 2000). Cloning and sequencing of 16S rRNA genes has been used to identify the presence of a diverse range of treponemes in polytreponemally infected tissue, but their highly fastidious nature has, for the most part, precluded any further characterization of these organisms. Although some progress has been made in elucidating the taxonomy of treponemes associated with human periodontal infections, only a single BDD-associated treponeme taxon, Treponema brennaborense (Schrank et al., 1999), has been proposed thus far. This is despite at least five treponeme phylotypes having been identified in BDD lesions (Choi et al., 1997) and several isolation reports of BDD treponemes that correspond to three of the five groups (Evans et al., 2008;Trott et al., 2003;Walker et al., 1995). Furthermore, T. brennaborense was not identified in any BDD lesions during a recent epidemiological study, suggesting that this species may not be as important in disease pathogenesis as initially perceived (Nordhoff et al., 2008).Abbreviations: BDD, bovine digital dermatitis; FCS, fetal calf serum.3Present address:

An epizootic in farmed, market-size rainbow trout in Spain caused by a strain of Carnobacterium piscicola of unusual virulence. Diseases ofAquatic Organisms 17,87-99 UBELS, J. L. & EDELHAUSER, H. F. (1987) Effects of corneal epithelial abrasion on corneal transparency, aqueous humor composition, and lens of fish.

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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.

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