Identification of candidate pathogens of papillomatous digital dermatitis in dairy cattle from quantitative 16S rRNA clonal analysis

Although it is suspected that papillomatous digital dermatitis (PDD), an infectious foot disease of cattle, is caused by multiple bacteria, it remains unclear precisely which ones are involved in the etiology. To study the bacterial community, we used 16S rRNA gene sequencing of randomly selected clones based on PCR with minimum amplification cycles to search for organisms present in PDD lesions but not in healthy foot skin. The nucleotide sequences of 1525 clones from 5 PDD lesions (836 clones) and 4 samples of healthy foot skin (689 clones) were determined and grouped into 316 operational taxonomic units (OTUs) with a cut-off value of >99% sequence identity. Two OTUs, P-01 (143 clones; 100% nucleotide sequence identity with Treponema phagedenis) and P-02 (112 clones; 86% identity with Bacteroidetes), were detected most frequently in all PDD samples examined. In contrast, OTU N-01 (87 clones), showing 99% nucleotide sequence identity with Moraxella phenylpyruvica, was the most prevalent in the normal samples examined. Spirochaetes were detected in only 1 sample. Phylogenetic analysis showed that T. denticola-like and T. phagedenis-like spirochetes were the predominant groups in the PDD lesions. Detection of multiple treponemes and an unknown bacterium close to Bacteroides sp. at high rates by a culture-independent approach could be evidence of the association of these organisms with PDD.