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.

A survey was made of 37 dairy farms in Wirral, mid-Cheshire, mid-Somerset and Dyfed, Wales, to assess the incidence and prevalence of lameness in the cows between May 1989 and September 1991. The incidence was obtained from records made whenever a cow was examined for lameness or received preventive foot-trimming. The mean annual incidence was 54.6 new cases per 100 cows with a range from 10.7 to 170.1 and the mean values during summer and winter were 22.9 and 31.7, respectively. The prevalence of lameness was measured by regular visits at which locomotion was scored on a scale of 1 to 5, and the prevalence of lameness was calculated for each visit as the proportion of cows with scores of 3 or more. The mean annual prevalence over the whole period was 20.6 per cent with a range from 2.0 to 53.9 per cent for the 37 farms. The mean prevalences during summer and winter were 18.6 and 25.0 per cent, respectively. The prevalence measured at a single visit in midsummer or midwinter was significantly correlated with the mean prevalence over the whole corresponding period and may be useful as an assessment of the extent of lameness in a herd and the efficacy of control measures. There was evidence that training farmers to recognise early cases of lameness and request veterinary treatment resulted in a marked reduction in the duration of cases of lameness.

Information from 37 dairy farms, in four regions of England and Wales provided data on 8991 lesions and the preventive trimming of 4837 cows' feet. Of the total of 13,828 forms returned, veterinary surgeons treated 32 per cent and farmers or stockmen 46 per cent. Of the 8645 lesions associated with episodes of lameness, lesions in the hindlimbs accounted for 92 per cent, of which 65 per cent were in the outer claw, 20 per cent in the skin and 14 per cent in the inner claw. Sole ulcers (40 per cent) and white line lesions (29 per cent) were the predominant diseases of horn, and digital dermatitis (40 per cent) was the most common disease of the skin. Subjective assessments showed that sandcrack, penetration of the sole by foreign bodies and interdigital necrobacillosis were associated with the most severe cases of lameness. There was a significant seasonal effect in the reporting of lesions.

This study aimed to isolate and characterize treponemes present in the bovine gastrointestinal (GI) tract and compare them with bovine digital dermatitis (BDD) treponemes. Seven spirochete isolates were obtained from the bovine GI tract, which, on the basis of 16S rRNA gene comparisons, clustered within the genus Treponema as four novel phylotypes. One phylotype was isolated from several different GI tract regions, including the omasum, colon, rumen, and rectum. These four phylotypes could be divided into two phylotype pairs that clustered closest with each other and then with different, previously reported rumen treponemes. The treponemes displayed great genotypic and phenotypic diversity between phylotypes and differed considerably from named treponeme species and those recently reported by metagenomic studies of the bovine GI tract. Phylogenetic inference, based on comparisons of 16S rRNA sequences from only bovine treponemes, suggested a marked divergence between two important groups. The dendrogram formed two major clusters, with one cluster containing GI tract treponemes and the other containing BDD treponemes. This division among the bovine treponemes is likely the result of adaptation to different niches. To further differentiate the bovine GI and BDD strains, we designed a degenerate PCR for a gene encoding a putative virulence factor, tlyC, which gave a positive reaction only for treponemes from the BDD cluster.Treponema species are typically anaerobic, fastidious, highly motile, spiral microorganisms and may be found in the oral cavity, digestive tract, and genital areas of humans, animals, and insects (20, 32). Several treponeme taxa are associated with disease, such as the human syphilis infectious agent Treponema pallidum (30), various Treponema species associated with human periodontal infections (10), and several treponeme phylotypes involved in bovine digital dermatitis (BDD) (4). In contrast, several treponemes have been reported to be commensal, living as symbionts in the gastrointestinal (GI) tracts of animals and insects. The spirochetes Treponema bryantii and Treponema saccharophilum have been isolated from the rumen of cows (26,36), and another three novel treponeme taxa have been isolated from GI material of pigs (6, 23). In insects, Treponema azotonutricium and Treponema primitia were isolated from the guts of termites (15).The relative paucity of data regarding treponemes and their locations in animal tissues is primarily the result of difficulties associated with their isolation, cultivation, and purification. More frequently, molecular techniques such as 16S rRNA gene clone libraries have been used to identify treponemes within animal samples but without subsequent culture. While no targeted studies of bovine GI tract treponeme 16S rRNA genes have been reported, several global bacterial studies showed that there are several phylotypes of treponemes in the bovine GI tract (11,29,38,44). Although only two bovine GI tract treponemes have been proposed as novel taxa (26, 36), considerable...