Molecular characterization of Treponema pallidum-related rabbit-derived material following experimental inoculation...

Syphilis remains a major sexually transmitted infection worldwide. Treponema pallidum subspecies pallidum is difficult to investigate under conventional laboratory cultivation conditions, which limits experimental and molecular studies involving clinical material. This pilot exploratory study aimed to obtain preliminary molecular evidence of Treponema pallidum-related rabbit-derived material following inoculation with human chancre-derived clinical specimens. Chancre-derived clinical material was examined by dark-field microscopy and used for experimental intratesticular inoculation in rabbits, followed by real-time polymerase chain reaction (PCR), conventional PCR targeting partial 16 S ribosomal RNA gene fragments, Sanger sequencing, and phylogenetic analysis.

Thin spiral-shaped structures morphologically consistent with treponemal organisms were observed in selected clinical specimens by dark-field microscopy. Real-time PCR demonstrated amplification signals consistent with Treponema pallidum-related DNA in three rabbit-derived samples following initial inoculation. Because inflammatory findings remained limited, these PCR-positive rabbits subsequently received secondary intratesticular inoculation using cryopreserved chancre-derived material as an exploratory modification intended to improve recovery of rabbit-derived material for downstream molecular analysis. Following secondary inoculation, these PCR-positive rabbits developed unilateral orchitis-like inflammatory changes involving the secondarily inoculated (contralateral) testis. Conventional PCR generated partial 16 S rRNA gene amplicons of the expected size (~ 1.5 kb) in PCR-positive samples. Sequencing analysis demonstrated high similarity to published Treponema pallidum reference sequences, with sequence identity ranging from approximately 99.45% to 100%. Phylogenetic analysis demonstrated clustering of the obtained sequences with published Treponema pallidum reference sequences retrieved from GenBank.

The combined use of dark-field microscopy, experimental rabbit inoculation, species-specific real-time PCR, sequencing, and phylogenetic analysis enabled preliminary molecular detection and partial sequence-based characterization of rabbit-derived Treponema pallidum-related material obtained following inoculation with chancre-derived clinical specimens. Although exploratory and limited by the small number of positive samples and partial 16 S ribosomal RNA gene analysis, the findings provide preliminary molecular evidence supporting the potential usefulness of rabbit-based experimental approaches for downstream molecular investigations.