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Final Program


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Basic & Translational Science

Category: Syphilis

(P385) Innovative refrigerated housing model for isolation of Treponema pallidum strains by rabbit infectivity testing in tropical climates.

Wednesday, July 26, 2023
16:00 – 18:00 CST


Introduction: Understanding the global phylogenetic diversity of Treponema pallidum (TPA), particularly in resource limited settings, is imperative for syphilis vaccine development. Intratesticular injection of clinical samples into New Zealand White rabbits (rabbit infectivity testing, RIT) is the only means to isolate TPA strains. The need for an animal research facility with strict temperature control poses a significant challenge for isolation of TPA in tropical cities, including Cali, a city in south-west Colombia with temperatures ranging between 20°C and 30°C (68°F to 86°F).





Methods: As part of a multicenter global consortium aiming to characterize the variability of TPA outer membrane proteins as potential syphilis vaccinogens, RIT was set-up in an animal research facility in Cali, Colombia. Initially rabbits were housed in cages in air-conditioned rooms. Rabbits (≥ 3 month-males‚ 3-4 Kg) were sedated intramuscularly and a 1500 mL of whole blood from early syphilis patients was injected in each testicle. Testes were extracted following seroconversion and TPA burdens were assessed by darkfield (DF) microscopy and quantification of polA copy numbers by qPCR.



Results: Between April 2020 and September 2021 TPA was identified in 2/12 infected rabbits housed in air-conditioned rooms with temperature set at 12°C. DF examination revealed low numbers of spirochetes and histopathology only minimal inflammation. Due to the high frequency of unsuccessful passage, all aspects of the RIT were revised identifying temperature fluctuations in the animal facility room with frequent temperatures up to 15°C during a 24-hour read. To address this problem, an industrial refrigerator with a controlled temperature set at 12°C was adapted to simultaneously house four rabbits in individual cubicles. Temperatures in the refrigerator remained stable between 10°C and 12°C during 24-hour reads. Rabbits appeared healthy and ate well. Since the implementation of this equipment, 6/12 rabbits have seroconverted with abundant TPA by DF and treponemal burdens well above the threshold (70 polA copies/µl) for whole genome sequencing.





Conclusion: Temperature control is critical aspect for rabbit isolation of TPA clinical strains. We developed a simple refrigerated housing system to recover TPA strains in regions where warm temperatures hinder the ability of animal facilities to maintain low temperatures.