Successful isolation of Treponema pallidum strains from patients' cryopreserved ulcer exudate using the rabbit model

Abstract

Clinical isolates of Treponema pallidum subspecies pallidum (T. pallidum) would facilitate study of prevalent strains. We describe the first successful rabbit propagation of T. pallidum from cryopreserved ulcer specimens. Fresh ulcer exudates were collected and cryopreserved with consent from syphilis-diagnosed patients (N = 8). Each of eight age-matched adult male rabbits were later inoculated with a thawed specimen, with two rabbits receiving 1.3 ml intratesticularly (IT), and six receiving 0.6 ml intravenously (IV) and IT. Monitoring of serology, blood PCR and orchitis showed that T. pallidum grew in 2/8 rabbits that were inoculated IV and IT with either a penile primary lesion specimen (CDC-SF003) or a perianal secondary lesion specimen (CDC-SF007). Rabbit CDC-SF003 was seroreactive by T. pallidum Particle Agglutination (TP-PA) and Rapid Plasma Reagin (RPR) testing, PCR+, and showed orchitis by week 6. Euthanasia was performed in week 7, with treponemal growth in the testes confirmed and quantified by qPCR and darkfield microscopy (DF). Serial passage of the extract in a second age-matched rabbit also yielded treponemes. Similarly, rabbit CDC-SF007 showed negligible orchitis, but was seroreactive and PCR+ by week 4 and euthanized in week 6 to yield T. pallidum, which was further propagated by second passage. Using the 4-component molecular typing system for syphilis, 3 propagated strains (CDC-SF003, CDC-SF007, CDC-SF008) were typed as 14d9f, 14d9g, and 14d10c, respectively. All 3 isolates including strain CDC-SF011, which was not successfully propagated, had the A2058G mutation associated with azithromycin resistance. Our results show that immediate cryopreservation of syphilitic ulcer exudate can maintain T. pallidum viability for rabbit propagation.

Fig 1. Schematic summarizing propagation of T. pallidum from patients’ lesion exudate specimens.

Each of eight rabbits was inoculated with one of the specimens listed, and the injection was either administered IT in the left testis, or both IV and IT (left testis) at the volumes indicated. Each rabbit was then monitored once weekly for up to three months until results indicated a positive infection status by the parameters shown, at which time euthanasia was performed. If propagation occurred, up to two serial passage(s) were performed using fresh or frozen extract from the previous passage rabbit. Rabbits that remained negative by all three measures of infection were euthanized at the three-month mark with no further passage.

Fig 2. Serology results for successfully propagated specimens.

Treponemal (TP-PA, black line, left axis) and nontreponemal (RPR, grey line, right axis) antibody titers in rabbits (N = 1 per passage) that were (A) inoculated IV and IT (0.6 ml inoculum per site) with the patient specimen CDC-SF003 and (B) inoculated IV and IT (0.6 ml inoculum per site) with the patient specimen CDC-SF007. Second passage was then performed for (C) CDC-SF003 inoculated IT with fresh testicular extract (1.5 ml) from the first passage rabbit and (D) CDC-SF007 inoculated IT with previously frozen and thawed testicular extract (1.5 ml) from the first passage rabbit. Whole blood was also analyzed weekly by PCR, with results shown in the grey bars for each time point.