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Deep sequencing analysis reveals temporal microbiota changes associated with development of bovine digital dermatitis.

ABSTRACT: Bovine digital dermatitis (DD) is a leading cause of lameness in dairy cattle throughout the world. Despite 35 years of research, the definitive etiologic agent associated with the disease process is still unknown. Previous studies have demonstrated that multiple bacterial species are associated with lesions, with spirochetes being the most reliably identified organism. This study details the deep sequencing-based metagenomic evaluation of 48 staged DD biopsy specimens collected during a 3-year longitudinal study of disease progression. Over 175 million sequences were evaluated by utilizing both shotgun and 16S metagenomic techniques. Based on the shotgun sequencing results, there was no evidence of a fungal or DNA viral etiology. The bacterial microbiota of biopsy specimens progresses through a systematic series of changes that correlate with the novel morphological lesion scoring system developed as part of this project. This scoring system was validated, as the microbiota of each stage was statistically significantly different from those of other stages (P < 0.001). The microbiota of control biopsy specimens were the most diverse and became less diverse as lesions developed. Although Treponema spp. predominated in the advanced lesions, they were in relatively low abundance in the newly described early lesions that are associated with the initiation of the disease process. The consortium of Treponema spp. identified at the onset of disease changes considerably as the lesions progress through the morphological stages identified. The results of this study support the hypothesis that DD is a polybacterial disease process and provide unique insights into the temporal changes in bacterial populations throughout lesion development.

SUBMITTER: Krull AC

PROVIDER: S-EPMC4136199 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Deep sequencing analysis reveals temporal microbiota changes associated with development of bovine digital dermatitis.

Krull Adam C AC Shearer Jan K JK Gorden Patrick J PJ Cooper Vickie L VL Phillips Gregory J GJ Plummer Paul J PJ

Infection and immunity 20140527 8

Bovine digital dermatitis (DD) is a leading cause of lameness in dairy cattle throughout the world. Despite 35 years of research, the definitive etiologic agent associated with the disease process is still unknown. Previous studies have demonstrated that multiple bacterial species are associated with lesions, with spirochetes being the most reliably identified organism. This study details the deep sequencing-based metagenomic evaluation of 48 staged DD biopsy specimens collected during a 3-year ...[more]

PMID: 24866801

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Project description:Bovine digital dermatitis (DD) is a skin disorder that is a significant cause of infectious lameness in cattle around the world. However, very little is known about the etiopathogenesis of the disease and the microbiota associated with DD in beef cattle. In this study, we provide a comprehensive characterization of DD and healthy skin microbiota of feedlot beef cattle. We also developed and validated a novel multiplex quantitative PCR (qPCR) assay to quantify the distribution of DD-associated bacterial species across DD lesion stages. We determined the DD-associated microbiota with deep amplicon sequencing of the V3-V4 hypervariable region of the 16S rRNA gene, followed by the application of novel and existing qPCR assays to quantify species distributions of Treponema, Porphyromonas, Fusobacterium, and Bacteroides across lesion stages. Deep amplicon sequencing revealed that Treponema, Mycoplasma, Porphyromonas, and Fusobacterium were associated with DD lesions. Culturing of DD biopsy specimens identified Porphyromonas levii, Bacteroides pyogenes, and two Fusobacterium spp. within DD lesions. Using species-specific qPCR on DD lesion DNA, we identified P. levii in 100% of active lesion stages. Early-stage lesions were particularly associated with Treponema medium, T. phagedenis, and P. levii. This study suggests a core DD microbial group consisting of species of Treponema, Fusobacterium, Porphyromonas, and Bacteroides, which may be closely tied with the etiopathogenesis of DD. Further characterizations of these species and Mycoplasma spp. are necessary to understand the microbial factors involved in DD pathogenesis, which will help elucidate DD etiology and facilitate more targeted and effective mitigation and treatment strategies. IMPORTANCE Previous work, primarily in dairy cattle, has identified various taxa associated with digital dermatitis (DD) lesions. However, there is a significant gap in our knowledge of DD microbiology in beef cattle. In addition, characterization of bacteria at the species level in DD lesions is limited. In this study, we provide a framework for the accurate and reproducible quantification of major DD-associated bacterial species from DNA samples. Our findings support DD as a polymicrobial infection, and we identified a variety of bacterial species spanning multiple genera that are consistently associated with DD lesions. The DD-associated microbiota identified in this study may be capable of inducing the formation and progression of DD lesions and thus should be primary targets in future DD pathogenesis studies.

Project description:At present, very little information exists regarding what role the environmental slurry may play as an infection reservoir and/or route of transmission for bovine digital dermatitis (DD), a disease which is a global problem in dairy herds. To investigate whether DD-related bacteria belong to the indigenous microbiota of the dairy herd environment, we used deep amplicon sequencing of the 16S rRNA gene in 135 slurry samples collected from different sites in 22 dairy farms, with and without DD-infected cows. Both the general bacterial populations and digital dermatitis-associated Treponema were targeted in this study. The results revealed significant differences in the bacterial communities between the herds, with only 12 bacterial taxa shared across at least 80% of all the individual samples. These differences in the herd microbiota appeared to reflect mainly between-herd variation. Not surprisingly, the slurry was dominated by ubiquitous gastrointestinal bacteria, such as Ruminococcaceae and Lachnospiraceae Despite the low relative abundance of spirochetes, which ranged from 0 to 0.6%, we were able to detect small amounts of bacterial DNA from DD-associated treponemes in the slurry. However, the DD-associated Treponema spp. were detected only in samples from herds with reported DD problems. These data indicate that treponemes involved in the pathogenesis of DD are not part of the normal environmental microflora in dairy herds without clinical DD and, consequently, that slurry is not a primary reservoir of infection.IMPORTANCE Bovine digital dermatitis (DD), a dermal disease which causes lameness in dairy cattle, is a serious problem worldwide. To control this disease, the infection reservoirs and transmission routes of DD pathogens need to be clarified. The dairy herd slurry may be a pathogen reservoir of DD-associated bacteria. The rationale for the present study was, therefore, to examine whether DD-associated bacteria are always present in slurry or if they are found only in DD-afflicted herds. The results strongly indicated that DD Treponema spp. are not part of the indigenous slurry and, therefore, do not comprise an infection reservoir in healthy herds. This study applied next-generation sequencing technology to decipher the microbial compositions of environmental slurry of dairy herds with and without digital dermatitis.

Dataset Information

Deep sequencing analysis reveals temporal microbiota changes associated with development of bovine digital dermatitis.

Publications

Deep sequencing analysis reveals temporal microbiota changes associated with development of bovine digital dermatitis.

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