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).