Treponema multi-locus sequence typing
In our previous work, we identified two variable gene loci for strain typing of TPE (tp0488 and tp0548 ) in nonhuman primates (Chuma et al., 2019), which we adapted for the use in our lagomorph samples. We note here the close genetic relationship betweenTPE and TP eC, which is over 98% based on the whole genome (Šmajs et al., 2011; Čejková et al., 2012), 97.8% for the tp0488gene and 90.3% for the tp0548 gene with all amplification primer binding sites being 100% conserved (Šmajs et al., 2011).
tp0488 : The PCR amplifies an ~830 bp region of the methyl-accepting chemotaxis protein 2 gene (mcp 2). We checked the previously published primers (Chuma et al., 2019) for compatibility to the published TP eC strain Cuniculi A (GenBank CP002103.1) and amplified the gene target as described previously (Chuma et al., 2019). Briefly, the 51 µl reaction volume comprised 45 µl Platinum PCR Super Mix High Fidelity (Thermo Fisher Scientific, Darmstadt, Germany), 2 µl of each 10 µM primer and template DNA, respectively. The amplification was performed using a SensoQuest Thermocycler (SensoQuest, Goettingen, Germany) applying the following conditions: two min pre-denaturation at 94°C followed by 80 cycles of 15 sec at 94°C, 15 sec at 59°C and 60 sec at 68°C.
tp0548 : We used a nested PCR to amplify a ~1,070 bp region of the tp0548 gene (encoding for a predicted outer membrane protein), using primers and cycling conditions as published elsewhere (Chuma et al., 2019) with the only exception of using a different polymerase. Briefly, the 50µl reaction solution contained 25 µl 2x Phanta Max Master Mix (Vazyme Biotech Co. Ldt., Nanjing, China), 19 µl RNase free water, 2 µl of the respective 10 µM primer and template DNA. For the nested PCR, we used 2 µl of the first PCR reaction. Cycling conditions for the first and nested PCR were 3 min initial denaturation at 95°C followed by 35 amplification cycles of 15 sec at 95°C, 15 sec at 48°C and an elongation phase at 72°C for 90 sec (first PCR) and 60 sec (nested PCR), respectively. Each PCR run ended with a post-extension step at 72°C for 5 min.