Introduction
Syphilis and yaws are caused by Treponema pallidum (TP ) ssp. pallidum (syphilis; TPA ) and ssp. pertenue(yaws; TPE ), respectively. While syphilis is a human disease with several hundred-year long history, yaws is a disease of humans and nonhuman primates (Majander et al., 2020; Beale et al., 2021). Studies on modern TPA lineages controversially discuss a common ancestor of all TPA strains in the 1700s (Arora et al., 2016). However, the evolution of TPA , its closest relative TPE and the genetically closely related lagomorph syphilis-causing bacteriumTreponema paraluisleporidarum ecovar Lepus (TP eL) in hares and T. paraluisleporidarum ecovar Cuniculus (TP eC) in rabbits remains elusive. Only a single complete genome of a rabbit-infecting laboratory-maintained strain from the USA has been whole-genome sequenced until today (Šmajs et al., 2011). Comparative studies showed that this strain, TP eC strain Cuniculi A, has a 98.1% whole-genome identity to the human syphilis-causing TPAstrain Nichols (Šmajs et al., 2011; Pětrošová et al., 2013). Although its genome is slightly smaller compared to that of the human infecting syphilis bacterium there is a general genome synteny (Šmajs et al., 2011). Yet, all information on genomic deletions, insertions or changes that likely code for the lagomorph host specificity is derived from the single published TP eC strain Cuniculi A genome (Strouhal et al., 2007; Šmajs et al., 2011). Consequently, the basis for human-pathogenicity cannot be identified unless more Treponemagenomes of lagomorph origin are analysed.
Our previous studies have demonstrated anti-Treponema antibodies and the presence of the bacterium in several European brown hare (Lepus europaeus ; EBH) populations (Nováková et al., 2019; Hisgen et al., 2020, 2021). Serology and quantitative PCR were, however, unable to distinguish infection with TP eL, TP eC or the genetically closely related TP . We note here that the latter is not known to infect wild lagomorphs, but rabbits are traditionally used to cultivate human treponemes in vivo which highlights the ability forTP to adapt and survive in the lagomorph host (Lukehart and Marra, 2007). In this study and prior to whole-genome sequencing, we were interested in the molecular epidemiology and the genetic diversity of treponemes infecting European lagomorphs. In the light of the previously reported wide-spread infection in the wild lagomorph-host, which argues for a well-established disease in European lagomorphs, we hypothesised that hare infecting strains have a strain diversity as high as the one seen in a comparative wild host-pathogen system – African nonhuman primates infected with the sister-bacterium TPE (Chuma et al., 2019). We predicted a geographic clustering of strains isolated from structurally connected hare populations. We used the naturally occurring Treponema infection in nonhuman primates as a direct comparison since the infection in wild lagomorphs is equally not under selection pressure from antibiotic treatment (Knauf et al., 2018) and has multiple host species involved. Due to the high genetic similarity of human and nonhuman primate infecting TP and hare and rabbit infecting TP eL/C (98.1% genome identity; (Šmajs et al., 2011)), we applied a multi-locus sequence typing system that was originally designed for TPE infection in nonhuman primates (Chuma et al., 2019).