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Abstract
Background Syphilis rates are again increasing in the US and globally and are associated with HIV transmission and rising rates of congenital syphilis. Humans serve as the only natural reservoir of the causative agent, Treponema pallidum, and no in vitro cultivation systems are available. Progress towards a vaccine and a better understanding of immune response to syphilis has been hampered over the last half century by lack of a murine model. We hypothesised that previous attempts to establish T pallidum infection in mice were unsuccessful because of rapid clearance of the organism by an intact innate immune response. Myd-88 serves as a common signalling molecule stimulated by most toll-like receptors TLRs; pattern recognition receptors of the innate immune system found on most innate immune cells, for example, monocytes, macrophages, and dentritic cells) and is responsible for downstream cytokine responses. We utilised mice bred to be Myd-88 deficient to test this hypothesis and to ascertain whether this mouse strain could be used to study persistent syphilis infection, immune response mechanisms, and eventually vaccine candidates.
Methods T pallidum, Nicohols strain, was cultivated by intratesticular infection of New Zealand male rabbits housed at 62°C as per protocol. After 12 day incubation, T pallidum was extracted and the concentration adjusted to 7×108 organisms/ml. Myd-88 −/− mice and equally-aged C57BL/6 mice were inoculated intradermally, intraperitoneally, intravaginally or in testicles, and intrarectally each mouse receiving total dose of 1×108 organisms divided into the four body site aliquots). Mice were observed daily for signs or cutaneous disease and/or systemic illness and were sacrificed at day 10 and 21. DNA and RNA were extracted from skin, spleen, genitals, rectum, lymph nodes, spinal cord and blood for use in real-time quantitative PCR and RT-PCR, respectively. Corresponding tissue types were also evaluated by histopathology and immunohistochemical staining T pallidum Ab, BioCare, Concord, California, USA). The experiment was repeated three times with variable number of mice per experiment.
Results A total of 18 Myd-88 mice and 19 C57BL/6 mice were infected. MyD-88 −/− mice were more likely than B6 control mice to have detectable RNA at day 10 and 21 (day 10, 12/35 sites (35%) +RNA in Myd-88 −/− ; 3/35 sites (8.5%) +RNA in WT. Day 21, 11/40 sites (27.5%) +RNA in Myd-88−/−; 3/40 (7.5%) +RNA in B6). One experiment ongoing past 42 day post-infection reveals at day 42 sacrifice Myd-88 −/− mice with 6/15 sites (40%) +RNA; B6 0/15 sites (0%) +RNA. Histopathology revealed mild to moderate inflammation in MyD88−/− mice and demonstrable organisms by IHC Abstract O4-S2.05 figure 1).
Day 21 Post T.pallidum infection Epididymis.
Conclusion These preliminary experiments suggest that the immune recognition impairment caused by deleting Myd88 signalling protein results in productive and longer-lasting T pallidum infection in this murine strain compared to wild-type mice. Further experiments are necessary to further elucidate the kinetics of T pallidum-infected Myd-88 −/− mice (relative DNA burden in tissue compartments, carrying out infection to 3 and 6 months and beyond to assess chronicity). MyD-88 deficient mice may hold the promise of serving as one of the first useful murine models to study immunopathogenesis of T pallidum infection. Abstract O4-S2.05 figure 1: representative epididymus sections from Day 21 sacrifice. Formalin-fixed tissues were stained with H&E as well as T pallidum-specific immunohistochemical stain (IHC).