Nicholas J. Evans

Three unique groups of spirochetes isolated from digital dermatitis lesions in UK cattle

Bovine digital dermatitis (BDD) is a severe infectious cause of lameness which has spread through dairy cattle populations worldwide, causing serious welfare and agricultural problems. Spirochetes are the main organisms implicated and have previously proven difficult to isolate. This study aimed to isolate and characterise the range of spirochetes associated with BDD in the UK. Twenty-three spirochete isolates were obtained from 30 BDD lesions, which by 16S rRNA gene and flaB2 gene analysis clustered within the genus Treponema as three phylogroups; groups 1 (Treponema medium/Treponema vincentii-like), 2 (Treponema phagedenis-like) and 3 (Treponema denticola/Treponema putidum-like). The treponemes displayed large genotypic and phenotypic diversity between phylogroups and differed from named treponeme species. A previously isolated contagious ovine digital dermatitis spirochete was located within one of the three phylogroups, group 3, and could also be identified within this group on the basis of phenotype testing, suggesting BDD and contagious ovine digital dermatitis may share the same aetiological agent. A strain isolated from a bovine interdigital dermatitis lesion, could be identified as part of BDD isolate group 2, suggesting bovine interdigital dermatitis and BDD may have the same causative agent. Two common enzyme activities, C4 esterase and C8 esterase lipase, were identified in all BDD associated treponemes suggesting common metabolic pathways for sharing this novel niche or even common virulence traits. Further studies are required to determine whether the three groups of novel treponemes are representative of new treponeme taxa and to delineate how they interact with bovine tissues to cause disease.

Association of Unique, Isolated Treponemes with Bovine Digital Dermatitis Lesions

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.

Association between bovine digital dermatitis treponemes and a range of 'non-healing' bovine hoof disorders

This study describes the association between bovine digital dermatitis (BDD) treponemes and three ‘non-healing’ bovine hoof horn lesions, namely, ‘toe necrosis’ (TN), ‘non-healing white line disease’ (nhWLD) and ‘non-healing sole ulcer’ (nhSU), which are disorders that involve penetration through the horn capsule to involve the corium. In this study, these non-healing disorders (n=44) were identified as foot lesions that exhibited a topical granular appearance, exuded a typical pungent smell, were severely painful to the animal involved, and typically originated from farms where BDD is endemic. Given the similarities between these ‘non-healing’ lesions and BDD, the authors subjected samples of diseased tissue to PCR assays to detect the presence of DNA of BDD treponemes. All the three characterised BDD treponeme groups were identified as present together in 84.2, 81.3 and 55.6 per cent of samples of TN (n=19), nhWLD (n=16) and nhSU (n=9), respectively. In contrast, healthy control horn samples from similar sites (n=16) were PCR-negative for the BDD treponemes. Hence, these non-healing hoof lesions were strongly associated with BDD treponemes. Samples from typical heel horn erosions (n=9) were also subjected to BDD treponeme PCR assays and no association could be identified between the BDD treponemes and this horn manifestation.

Identification of Spirochetes Associated with Contagious Ovine Digital Dermatitis

ABSTRACT Spirochetes of the genus Treponema were cultured from 7 of 10 cases of digital dermatitis in sheep. Two cultures comprised Treponema phagedenis-like and Treponema medium/Treponema vincentii-like spirochetes, respectively, while the remaining cultures comprised mixed populations of Treponema medium/Treponema vincentii-like, Treponema phagedenis-like, and Treponema denticola/Treponema putidum-like organisms.

Treponema pedis sp. nov., a spirochaete isolated from bovine digital dermatitis lesions

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) (=DSM 18691(T)=NCTC 13403(T)).

Characterization of novel bovine gastrointestinal tract Treponema isolates and comparison with bovine digital dermatitis treponemes.

ABSTRACT 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.

Detection and Molecular Characterization of Escherichia coli CTX-M-15 and Klebsiella pneumoniae SHV-12 β-Lactamases from Bovine Mastitis Isolates in the United Kingdom

ABSTRACT Recent reports raised concerns about the role that farm stock may play in the dissemination of extended-spectrum β-lactamase (ESBL)-producing bacteria. This study characterized the ESBLs in two Escherichia coli and three Klebsiella pneumoniae subsp. pneumoniae isolates from cases of clinical bovine mastitis in the United Kingdom. Bacterial culture and sensitivity testing of bovine mastitic milk samples identified Gram-negative cefpodoxime-resistant isolates, which were assessed for their ESBL phenotypes. Conjugation experiments and PCR-based replicon typing (PBRT) were used for characterization of transferable plasmids. E. coli isolates belonged to sequence type 88 (ST88; determined by multilocus sequence typing) and carried blaCTX-M-15 and blaTEM-1, while K. pneumoniae subsp. pneumoniae isolates carried blaSHV-12 and blaTEM-1. Conjugation experiments demonstrated that blaCTX-M-15 and blaTEM-1 were carried on a conjugative plasmid in E. coli, and PBRT identified this to be an IncI1 plasmid. The resistance genes were nontransferable in K. pneumoniae subsp. pneumoniae isolates. Moreover, in the E. coli isolates, an association of ISEcp1 and IS26 with blaCTX-M-15 was found where the IS26 element was inserted upstream of both ISEcp1 and the blaCTX-M promoter, a genetic arrangement highly similar to that described in some United Kingdom human isolates. We report the first cases in Europe of bovine mastitis due to E. coli CTX-M-15 and also of bovine mastitis due to K. pneumoniae subsp. pneumoniae SHV-12 β-lactamases in the United Kingdom. We also describe the genetic environment of blaCTX-M-15 and highlight the role that IncI1 plasmids may play in the spread and dissemination of ESBL genes, which have been described in both human and cattle isolates.

Host and environmental reservoirs of infection for bovine digital dermatitis treponemes

Bovine digital dermatitis (BDD) is a global infectious disease causing lameness of cattle and is responsible for substantial animal welfare issues and economic losses. The causative agents are considered to be spirochetal bacteria belonging to the genus Treponema, which have consistently been identified in BDD lesions worldwide. One potential means of controlling infection is the disruption of transmission; however, the infection reservoirs and transmission routes of BDD treponemes have yet to be elucidated. To address these issues, we surveyed for evidence of BDD treponeme presence in the dairy farm environment, in bovine tissues and in bovine gastrointestinal (GI) tract contents. A total of 368 samples were tested using PCR assays specific for each of three currently recognised, isolated phylotypes of BDD treponemes. All environmental samples, together with insects and GI tract content samples were negative for BDD treponeme DNA from the three phylotypes. However, we identified BDD treponemes in two non-pedal bovine regions: the oral cavity (14.3% of cattle tested) and the rectum (14.8% of cattle tested). Whilst only single phylotypes were detected in the oral cavity, two of the rectal tissues yielded DNA from more than one phylotype, with one sample yielding all three BDD treponeme phylotypes. Whilst it might be considered that direct skin to skin contact may be a major transmission route of BDD treponemes, further studies are required to characterise and determine the potential contribution of oral and rectal carriage to BDD transmission.

Contagious ovine digital dermatitis: An emerging disease.

The novel sheep disease, contagious ovine digital dermatitis (CODD) was first described in the UK in 1997. The disease is characterised by severe lameness associated with initial inflammation at the coronary band, followed by progressive separation of the hoof capsule from the underlying tissue. On microbiological examination, treponeme bacteria have been frequently isolated from cases of CODD, including treponemes phylogenetically identical to those associated with bovine digital dermatitis (BDD). Dichelobacter nodosus and Fusobacterium necrophorum have also been isolated from CODD lesions although their role in the pathogenesis remains uncertain. While epidemiological data indicate that the prevalence of CODD is increasing in the UK, the routes of transmission and associated risk factors have not been clearly elucidated. Evidenced-based treatment trials indicate that parenteral administration of long-acting amoxicillin is an efficacious treatment for CODD, while anecdotal evidence suggests other antibiotics, given locally and/or parenterally, may also be beneficial. Further microbiological and epidemiological research is urgently required to develop sustainable control strategies, including the development of vaccines and appropriate biosecurity and farm management protocols. In this review current knowledge of the clinical, aetiological, and epidemiological aspects of CODD is assessed as well as approaches to its control.

Association of treponemes with bovine ulcerative mammary dermatitis

Bovine ulcerative mammary dermatitis (UMD), also known as mammary necrotic dermatitis, udder seborrhoea, intertrigo or foul udder, presents as lesions on the udder in dairy cows ([Blowey and Weaver 1990][1], [Beattie and Taylor 2000][2], [Stamm and others 2009][3]). The typical UMD site is between