Treponema paraluisleporidarum multi-locus sequence
typing
We obtained positive PCR results from 405 samples from the tp0488gene (Germany: n=343/938;, Sweden: n=4/4; England: n=12/25; Italy:
n=14/81; the Netherlands: n=18/32; Czech Republic: n=14/15) and 439
sequences from the tp0548 gene (Germany: n=380/938;, Sweden:
n=4/4; England: n=13/25; Italy: n=14/81; the Netherlands: n=13/32; Czech
Republic: n=15/15; Table S1). In 57 samples we could only amplify thetp0488 gene and in 91 samples we were only able to amplify thetp0548 gene target. A total of 349 sequences (tp0488 = 195
and tp0548 = 154) had to be excluded from analysis due to low
sequence quality or high background noise due to superimposed sequences.
This resulted in 212, 296 and 194 sequences for tp0488 ,tp0548 and the concatenated gene target sequences, respectively,
including the reference sequence from the TP eC strain Cuniculi A
(CP002103.1; locus tags TPCCA_RS02365 and TPCCA_RS02685 )
and T. pallidum ssp. endemicum strain Iraq B (CP032303.1;
locus tags TENDIB_0488 and TENDIB_0548). We generated a
maximum-likelihood tree based on the concatenated sequences of thetp0488 and tp0548 genes and added the geographic origin as
attributes, with samples grouped into Northern (Schleswig Holstein and
northern part of Lower Saxony), Central and Western (southern part of
Lower Saxony, Hesse and North Rhine-Westphalia) and Southern Germany
(Bavaria and Baden-Wuerttemberg) as well as the Netherlands, Italy,
Czech Republic and the United Kingdom. Overall, there is no clustering
of the samples according to their geographic origin (Figure 2). While
bootstrap support for nodes in the phylogenetic tree is generally low,
there are some significantly supported distinctive features that are
noteworthy. The tree exhibits an initial split into two clades of which
one contains sequences obtained from EBHs sampled in Baden-Wuerttemberg
and Bavaria (Southern Germany) and one EBH from North Rhine-Westphalia
(Western Germany). In addition, the clade contains the TPe C
reference strain Cuniculi A and three strains of mountain hares from
Sweden. A sample from the fourth Swedish mountain hare (V1313_03_L1)
is found in the second clade and clusters together with all other EBH
samples as well as a strain that was found in a pet rabbit in Hesse
(Central Germany). The latter is identical to a strain obtained from a
EBH from Lower Saxony, approximately 140 km from the pet rabbit sampling
location. Within both main clusters, a number of statistically supported
subclades of geographically related samples e.g., from the Czech
Republic or Northern Germany, were found.
In our analysis we identified positively selected sites (codons) in each
of the target genes including tp0488 (n = 29) and tp0548(n = 54) (Table S1). Those sites were removed from the alignments and
only non-positively selected parsimony-informative sites as well as
singletons were used for network constructions. The minimum spanning
network that resulted from non-positively selected single nucleotide
variants within the tp0548 locus (Figure S1) does not change the
overall topology of the maximum-likelihood tree shown in Figure 2 and
equally lacks the overall geographic clustering of the samples.
Maximum-likelihood trees for individual loci can be found in the
Supplemental Material (Figure S2 and S3).
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Add Figure 2 about here
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