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Evidence of multiple Treponema phylotypes involved in bovine digital dermatitis as shown by 16S rRNA gene analysis and fluorescence in situ hybridization.

ABSTRACT: The etiopathogenesis of the skin disease digital dermatitis (DD), an important cause of lameness in cattle, remains uncertain. Microscopically, the disease appears to be polymicrobial, with spirochetes as the predominant bacteria. The objective of this study was to identify the main part of the bacteria involved in DD lesions of cattle by using culture-independent molecular methods. Ten different phylotypes of Treponema were identified either by 16S rRNA gene sequencing of bacteria from DD lesions or by fluorescence in situ hybridization (FISH) analysis using phylotype-specific 16S rRNA-directed oligonucleotide probes. Two phylotypes, phylotype 1 (PT1) and PT2, were not closely related to any characterized treponemal species. PT7 was 99.3% identical to Treponema denticola, while PT9 resembled T. vincentii by 96%. The remaining phylotypes, PT3, PT4, PT5, PT6, and PT8, and Treponema brennaborense had previously been isolated from DD lesions. Forty DD biopsy specimens were examined for Treponema by FISH. With one exception, all of the biopsy specimens revealed epidermotropic, intermingled infection with three or more different phylotypes (mean, 4.7). The most prevalent species were PT1 (95%), PT6 (93%), and PT3 (85%). While colonization by PT3 was confined to the surface of the epidermis, both PT1 and PT6 invaded deep into the stratum spinosum and were seen in ulcerated dermal papillae. In two cases, all 10 phylotypes were demonstrated. Furthermore, FISH with a Treponema group-specific probe showed that Treponema accounted for more than 90% of the total bacterial population in the biopsy specimens. These data strongly suggest that a group of apparently symbiotic Treponema species are involved as primary bacterial pathogens in DD.

SUBMITTER: Klitgaard K

PROVIDER: S-EPMC2546760 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Evidence of multiple Treponema phylotypes involved in bovine digital dermatitis as shown by 16S rRNA gene analysis and fluorescence in situ hybridization.

Klitgaard Kirstine K Boye Mette M Capion Nynne N Jensen Tim K TK

Journal of clinical microbiology 20080618 9

The etiopathogenesis of the skin disease digital dermatitis (DD), an important cause of lameness in cattle, remains uncertain. Microscopically, the disease appears to be polymicrobial, with spirochetes as the predominant bacteria. The objective of this study was to identify the main part of the bacteria involved in DD lesions of cattle by using culture-independent molecular methods. Ten different phylotypes of Treponema were identified either by 16S rRNA gene sequencing of bacteria from DD lesio ...[more]

PMID: 18562583

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Project description:Abortion in cattle causes significant economic losses for cattle farmers worldwide. The diversity of abortifacients makes abortion diagnostics a complex and challenging discipline that additionally is restrained by time and economy. Microbial culture has traditionally been an important method for the identification of bacterial and mycotic abortifacients. However, it comes with the inherent bias of favoring the easy-to-culture species, e.g., those that do not require cell culture, pre-enrichment, a variety of selective growth media, or different oxygen levels for in vitro growth. Molecular methods such as polymerase chain reaction (PCR) and next-generation sequencing have been established as alternatives to traditional microbial culturing methods in several diagnostic fields including abortion diagnostics. Fluorescence in situ hybridization (FISH), a bridging microscopy technique that combines molecular accuracy with culture independence, and spatial resolution of the pathogen-lesion relation, is also gaining influence in several diagnostic fields. In this study, real-time quantitative PCR (qPCR), 16S rDNA amplicon sequencing, and FISH were applied separately and in combination in order to (i) identify potentially abortifacient bacteria without the bias of culturability, (ii) increase the diagnostic rate using combined molecular methods, (iii) investigate the presence of the difficult-to-culture zoonotic agents Coxiella burnetii, Chlamydia spp., and Leptospira spp. in bovine abortions in Denmark. Tissues from 162 aborted or stillborn bovine fetuses and placentas submitted for routine diagnostics were screened for pathogenic bacteria using 16S rDNA amplicon sequencing. Lesion association of fungal elements, as well as of selection of bacterial abortifacients, was assessed using specific FISH assays. The presence of Chlamydia spp. and chlamydia-like organisms was assessed using qPCR. The study focused on bacterial and fungal abortifacients, because Danish cattle is free from most viral abortifacients. The 16S rDNA amplicon sequencing-guided FISH approach was suitable for enhancing abortion diagnostics, i.e., the diagnostic rate for cases with tissue lesions (n = 115) was increased from 46 to 53% when compared to routine diagnostic methods. Identification of Bacillus licheniformis, Escherichia coli, and Trueperella pyogenes accounted for the majority of additional cases with an established etiology. No evidence for emerging or epizootic bacterial pathogens was found. The difficult-to-culture abortifacients were either not detected or not identified as abortifacients.

Dataset Information

Evidence of multiple Treponema phylotypes involved in bovine digital dermatitis as shown by 16S rRNA gene analysis and fluorescence in situ hybridization.

Publications

Evidence of multiple Treponema phylotypes involved in bovine digital dermatitis as shown by 16S rRNA gene analysis and fluorescence in situ hybridization.

Similar Datasets

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