Shotgun Metagenomic Sequencing Reveals Functional Genes and Microbiome Associated with Bovine Digital Dermatitis

Abstract

Metagenomic methods amplifying 16S ribosomal RNA genes have been used to describe the microbial diversity of healthy skin and lesion stages of bovine digital dermatitis (DD) and to detect critical pathogens involved with disease pathogenesis. In this study, we characterized the microbiome and for the first time, the composition of functional genes of healthy skin (HS), active (ADD) and inactive (IDD) lesion stages using a whole-genome shotgun approach. Metagenomic sequences were annotated using MG-RAST pipeline. Six phyla were identified as the most abundant. Firmicutes and Actinobacteria were the predominant bacterial phyla in the microbiome of HS, while Spirochetes, Bacteroidetes and Proteobacteria were highly abundant in ADD and IDD. T. denticola-like, T. vincentii-like and T. phagedenis-like constituted the most abundant species in ADD and IDD. Recruitment plots comparing sequences from HS, ADD and IDD samples to the genomes of specific Treponema spp., supported the presence of T. denticola and T. vincentii in ADD and IDD. Comparison of the functional composition of HS to ADD and IDD identified a significant difference in genes associated with motility/chemotaxis and iron acquisition/metabolism. We also provide evidence that the microbiome of ADD and IDD compared to that of HS had significantly higher abundance of genes associated with resistance to copper and zinc, which are commonly used in footbaths to prevent and control DD. In conclusion, the results from this study provide new insights into the HS, ADD and IDD microbiomes, improve our understanding of the disease pathogenesis and generate unprecedented knowledge regarding the functional genetic composition of the digital dermatitis microbiome.

Fig 1. Phyla abundances ordered from the most abundant to the least abundant by HS, ADD and IDD.

Only the top 12 most abundant phyla are shown. The y-axis represents the mean abundances of annotation in each phylum on a log scale. Error bars are standard error of the mean. Different letters means P < 0.05.

Fig 2. MG-RAST heatmap depicting phyla distribution from ADD, IDD and HS.

Red and green colors scale represents low and high abundance, respectively.

Fig 3. Normalized abundance of Treponema spp. from HS, ADD and IDD.

Error bars are standard error of the mean. *P < 0.05.

Fig 4. MG-RAST heatmap displaying function distribution from ADD, IDD and HS.

Red and green colors scale represents low and high abundance, respectively.

Fig 5. Normalized abundance of genes related with flagellar motility in HS, ADD and IDD.

Error bars are standard error of the mean. Different letters means P < 0.05.

Fig 6. Bubble plot representing differences in abundance of flagellum associated proteins between lesions and healthy skin (Fig 7A: active lesion vs. healthy skin and Fig 7B: inactive lesion vs. healthy skin).

The Y axis represents the robust LogWorth of the false discovery rate and the X axis represents the percentage of prevalence when comparing healthy skin to lesions stages. The size of the circles denotes the effect size. Red line represents P < 0.00005.

Fig 7. Normalized abundance distribution of resistance to antibiotics and toxic compounds of HS, ADD and IDD.

Error bars represent standard error of the mean. Different letters means P < 0.05.

Fig 8. Recruitment plot for ADD (samples 4567749.3, 4567750.3, 4567751.3, 4567752.3) mapped on T. denticola ATCC 35405, T. vincentii ATCC 35580 and T.pallidum subsp. pallidum str. Nichols.

The blue circle represents the bacterial contigs for the genome of interest, while the two black rings map genes on the forward and reverse strands. Bars represent hits distributions and the colors are coded according to the e-value of the matches with red (-30 and less), orange (-20 to -30), yellow (-10 to -20), green (-5 to -10) and blue (-3 to -5).

Fig 9. Recruitment plot for IDD (samples 4567761.3, 4567762.3 and 4567763.3) mapped on T. denticola ATCC 35405, T. vincentii ATCC 35580 and T.pallidum subsp. pallidum str. Nichols.

The blue circle represents the bacterial contigs for the genome of interest, while the two black rings map genes on the forward and reverse strands. Bars represent hits distributions and the colors are coded according to the e-value of the matches with red (-30 and less), orange (-20 to -30), yellow (-10 to -20), green (-5 to -10) and blue (-3 to -5).