Rabbit intradermal inoculations as an efficient method to quantitate Treponema pallidum

Background. Histological examination and rabbit intratesticular inoculation of clinical specimens are traditionally used to diagnose syphilis and characterize the aetiological agent, Treponema pallidum. Usually, these methods provide only qualitative data of the bacterium. For quantitative analysis, dark-field microscopy and real-time PCR are commonly used but both methods have important limitations. We investigated intradermal inoculations in rabbits as a sensitive and quantitative alternative test method.Methods. We prepared 10-fold serial dilutions of a treponemal stock ranging from 4×10³ to 4 treponemes ml^-1. Each dilution series was injected intradermally in two rabbits, ten sites per rabbit. Similarly, each dilution was inoculated intratesticularly into two rabbits, four dilutions in a total of eight rabbits. We scored and measured the size of lesions at the injection sites in rabbits inoculated intradermally. We monitored intratesticularly infected rabbits for orchitis. Sera from all animals were tested for seroconversion. We also compared the analytical sensitivity of animal bioassays to that of treponemal PCR.Results. The infectivity titre of T. pallidum was the same whether measured by intradermal or intratesticular inoculations, but the former used 75% fewer animals. Bacterial concentration at 50% endpoint was lower, 4 treponemes ml^-1, with intratesticular route compared to 25 treponemes ml^-1 using intradermal injections. Nested PCR demonstrated an analytical sensitivity of 40 treponemes ml^-1, comparable to that obtained with animal bioassays. We also showed a correlation between lesion size and treponemal counts that, under standardized conditions, might be exploited to estimate the number of treponemes in a sample.Conclusions. Our studies showed that all methods tested detected T. pallidum with similar sensitivities. However, the intradermal route might be better suited to investigate clinical specimens as it offered desirable features such as treponemal quantification, minimal number of animals required and detection of live infectious bacteria, all in a single test.