Discussion
We show further evidence of TP eC/L infection in wild lagomorphs in Europe based on DNA isolated from swab and tissue material. This complements our previous serological and qPCR findings that already suggested a widespread infection in European lagomorphs (Nováková et al., 2019; Hisgen et al., 2020, 2021). Moreover, we confirm infection in three previously reported host species (EBH, mountain hare and rabbits) and a newly identified host, the Corsican hare. Unfortunately, in this study, we cannot make a statement about prevalence rates which is particularly the case for the two Corsican hares samples that are included into our sample set. Reports about hybridization in hare species (Pierpaoli et al., 1999; Marques, Farelo et al., 2017; Seixas et al., 2018; Pohjoismäki et al., 2021) highlight feasible transmission pathways for the interspecies spread of Treponema . In this context, the demonstrated infection of a pet rabbit and a EBH from Central Germany with an identical strain (01GIF22900120 and 02DNTWF1100120, Figure 1) is more difficult to explain since a direct interaction of the pet rabbit and wild hares can be excluded. Whole genome sequencing of the two strains is needed to prove whether the twoTP eC/L isolates are truly identical. To our knowledge, sexual interactions of rabbits and hares – even under natural conditions – has not been reported. In primate infection (including humans) with the related bacterium T. pallidum subsp. Pertenue , vector transmission through flies has been discussed (Knauf et al., 2016; Houinei et al., 2017; Gogarten et al., 2019). In lagomorphs vector transmission of Tp eC/L has not been investigated.
Our current data add novel insight into the genetic diversity of lagomorph infecting Treponema from Italy, Czech Republic, Germany, Sweden, the Netherlands and the United Kingdom (Figure 1). We used our established MLST system for nonhuman primate yaws infection (Chuma et al., 2019) to equally characterise lagomorph infecting strains. This was done under the assumption that the lagomorph infectingTP eC/L and T. pallidum are closely related as shown on the basis of the single published whole genome of TP eC (Strouhal et al., 2007; Šmajs et al., 2011). The results presented in Figure 2, demonstrate an unexpectedly greater diversity compared to what we have seen in non-treated naturally yaws infected nonhuman primates in sub-Saharan Africa. In the case of lagomorph tp0548 sequences, 242 variants – including differences in the length of repetitions – out of 295 obtained sequences were found, demonstrating the enormously high degree of genetic variability. Yet, most variable nucleotides in both loci, tp0488  and tp0548 , were found under positive selection, partly explaining the huge observed genetic diversity and the lack of geographical clustering. Interestingly, a human syphilis multilocus sequence typing system also uses partial analysis of thetp0548 gene for molecular typing of clinical isolates. Until now, 77 different alleles of tp0548 in TP have been identified from a total of 944 investigated clinical isolates (Grillová et al. 2019), suggesting that similar evolutionary forces operate on the tp0548  locus in human and lagomorph infecting Treponema . The higher observed genetic diversity of tp0548  in lagomorphs could be explained by prolonged infection in hares than in humans who are treated with antibiotics. It is open to debate whether the higher genetic diversity of lagomorph infectingTP eC/L mimics treponemal evolution in an untreated human population.
The strain diversity, geographic range of infection and the involvement of multiple lagomorph species are all indicators for the endemic character of the disease in European lagomorphs. In contrast to nonhuman primates (Chuma et al., 2019) and human infections (Beale et al., 2021) with the sister bacterium T. pallidum , our current data from lagomorphs showed only weak geographic clustering. This is unexpected, since the biology of hares – a mostly philopatric species that shows only limited dispersal activity (Avril et al., 2011) – would likely contribute to the long-term circulation of regional (dominant) strains in the different hare metapopulations. It is open to debate whether this is the result of the positive selection of variants in the gene targets that we used for molecular typing or an effect of the anthropogenic influence on the population through trans- or relocation (Masseti and Marinis, 2008; Sokos et al., 2015; Sánchez-García et al., 2021). Until today, the management of overexploited EBH populations is based on annual restocking (Canu et al., 2013). In combination with the mt-genome data (Figure S5), which indicate a panmictic EBH population, it is most likely that EBH dispersal and associated with this, the dispersal ofTP eL strains, is dominated by human influence.
In TP eC/L, the tp0548 locus shows not only a higher number of nucleotide variants compared to nonhuman primate and human infection with TPE and TPA , respectively, but also various types of short repeat units that have not been described in primate treponematoses (Figure 3). While the functional aspect of thesetp0548 short repeat units remains subject to ongoing investigations, it is likely that it enables the pathogen to better survive in its lagomorph host. From an evolutionary perspective, short, repeated nucleotide sequences in bacteria are frequently associated with higher replication error rates caused by slipped-strand mispairing (Deitsch et al., 2009; Castillo-Lizardo et al., 2014). It seems obvious, that a provoked slipped-strand mispairing in structurally non-essential parts of antigenic outer-membrane proteins provides an advantage over spontaneous mutations in terms of immune escape.
Apparently, the current selected loci – tp0488 and in particulartp0548 – are not well suited for the molecular typing of treponemes of lagomorph origin. This is reflected by the high number of haplotypes. Once a reasonable number of whole genome sequences ofTP eC/L becomes available, an in-depth revision of the current typing system is necessary to include more decently variable loci that are suitable for the epidemiological monitoring of transmission chains. Moreover, whole genome sequencing of modern and ancient samples from a wide geographic range and from multiple lagomorph species, including those that are not yet investigated (broom hare (L. castroviejoi ) in northern Spain, the cape hare (L. capensis ) in Sardinia and Cyprus, the Iberian hare (L. granatensis ) on the Iberian peninsula) will help backtrack the evolutionary path of the pathogen and its relationship to modern syphilis in humans
We have demonstrated the presence of TP eC/L using nucleic amplification assays and subsequent Sanger sequencing. These methods prevent us from making a final statement on the viability of the treponemes. Yet, in the authors’ view, the consistence of infection in lagomorphs sampled across Europe and the high copy numbers detected in samples of the internal genital of EBHs (Hisgen et al., 2021) makes an active infection likely. In human syphilis, with exception of latent syphilis, active infection is associated with clinical lesions (Lukehart, 2008), which was rarely seen in the clinically inspected lagomorphs, of which 20/532 (3,8%) had typical skin lesions. Unfortunately, many samples included into this study originated from collaborating hunting parties which limited the clinical expectation of the integument, the oral-cavity and the genital tract during the sampling procedure. In these samples, it cannot be excluded that lesions have been overlooked for example when the ulcer was located in the urethra as it is described for humans (Chambers et al., 2018).