The Applied Development of a Tiered Multilocus Sequence Typing (MLST) Scheme for Dichelobacter nodosus

Abstract

Dichelobacter nodosus (D. nodosus) is the causative pathogen of ovine footrot, a disease that has a significant welfare and financial impact on the global sheep industry. Previous studies into the phylogenetics of D. nodosus have focused on Australia and Scandinavia, meaning the current diversity in the United Kingdom (U.K.) population and its relationship globally, is poorly understood. Numerous epidemiological methods are available for bacterial typing; however, few account for whole genome diversity or provide the opportunity for future application of new computational techniques. Multilocus sequence typing (MLST) measures nucleotide variations within several loci with slow accumulation of variation to enable the designation of allele numbers to determine a sequence type. The usage of whole genome sequence data enables the application of MLST, but also core and whole genome MLST for higher levels of strain discrimination with a negligible increase in experimental cost. An MLST database was developed alongside a seven loci scheme using publically available whole genome data from the sequence read archive. Sequence type designation and strain discrimination was compared to previously published data to ensure reproducibility. Multiple D. nodosus isolates from U.K. farms were directly compared to populations from other countries. The U.K. isolates define new clades within the global population of D. nodosus and predominantly consist of serogroups A, B and H, however serogroups C, D, E, and I were also found. The scheme is publically available at https://pubmlst.org/dnodosus/.

Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications

“…Microreact is a web tool developed to produce these visualisations and the site provides a means for data to be automatically uploaded from other resources, such as PubMLST 62 . This can be demonstrated using data from a recent paper describing the development of a MLST scheme for Dichelobacter nodosus , the causative agent of ovine footrot, that indicated that the global bacterial population was geographically structured 36 . All isolates that included a genome record in the D. nodosus PubMLST database (n=171) were selected and the Microreact analysis run using cgMLST loci.…”

Genomic heterogeneity of Dichelobacter nodosus within and between UK sheep flocks and between age groups within a flock

“…The bacterial isolation, DNA isolation, sequencing and analysis of sequence data in this study are as described previously in (Blanchard et al, 2018). A total of 2,126 Interdigital swabs (E-swabs 480CE, Copan U.S.A.) were taken from ewes and lambs.…”

“…As described by (Blanchard et al, 2018), DNA was isolated using the Qiagen Cador Pathogen Mini Kit, following the manufacturers guidelines and eluted in 60 µl of elution buffer. DNA was sent to MicrobesNG (Birmingham University, U.K.), for sequencing using the Illumina MiSeq at 2 × 250 bp [Raw data is available in the Short Read Archive (PRJNA386733)].…”

“…Sequence reads were assembled using the A5-MiSeq pipelines, (Blanchard et al, 2018) and (Coil et al, 2015). The raw reads were analysed for overall quality and sequence adaptors using trimmomatic (Bolger et al, 2014).…”

“…In silico serogroup determination was completed using the fimA serogroup PCR primers developed by (Zhou et al, 2001) and phenotypic (aprV2/aprB2) determination made use of the PCR primers created by (Frost et al, 2015). For sequence type determination and cgMLST analysis the isolate data was uploaded to the D. nodosus MLST database (https://pubmlst.org/dnodosus/) (Blanchard et al, 2018).…”