Characterization of the isolate
The isolate was serologically characterized using polyclonal antibodies
according to previous recommendation (Dikken and Kmety, 1978). A panel
of 34 specific antisera representing 28 serogroups was used (Andamana,
Australis, Autumnalis, Ballum, Bataviae, Canicola, Celledoni, Codice,
Cynopteri, Djasiman, Grippotyphosa, Hebdomadis, Holland,
Icterohaemorrhagiae, Javanica, Louisiana, Lyme, Manhao, Mini, Panama,
Pomona, Pyrogenes, Ranarum, Sarmin, Sejroe, Semaranga, Shermani and
Tarassovi), and serogroup identification was confirmed by the
identification of the highest titration against specific representative
serovars included in the panel.
For molecular characterization, DNA from the isolated strain was
extracted using Illustra Bacterium Genomic Prep Mini Spin kit following
the manufacturer’s instructions (GE Healthcare, Sao Paulo, SP, Brazil).
The complete genome was sequenced through an Ion Torrent Personal Genome
Machine (PGM) (Life Technologies, Saint Aubin, France) sequencing
platform, using a 316 chip (Life Technologies) and single-end libraries
to generate readings of 400 bpon the BAM format, which were then
converted to the FASTQ format with the bamTofastq tool from the BEDTools
toolset. For the identification of Leptospira species, the
generated contigs were aligned using BLAST against the Multilocus
Sequence Typing (MLST) sequence sets proposed elsewhere (Boonsilp et
al., 2013). The allelic profile and sequence type obtained (ST) was
compared to other publicly available sequences from the PubMLST
(http://pubmlst.org/leptospira).