Ez1 apparatus

Fourteen Borrelia spp. were grown at 32°C in Barbour-Stoenner-Kelly-H (BSK-H) medium (Sigma, Saint Quentin Fallavier, France) supplemented with 10% heat-inactivated rabbit serum (Eurobio, Courtaboeuf, France) (Table 1). Dark-field microscopic observation was performed to ensure the absence of any contaminants and to verify the richness and viability of the culture. The identification of the growing borreliae was performed by PCR-sequencing the flagellin gene, as previously reported [19] . The Borrelia culture was centrifuged at 13,000 g for 10 min at room temperature, and the pellet was washed twice with 1 mL high performance liquid chromatography grade water (VWR International, Fontenay-sous-Bois, France) and then suspended at 10⁴ spirochetes/mL in this water before MALDI-TOF-MS analysis.
Fifty ticks were collected in Senegal. Total DNA was extracted from the body of the tick by using the EZ1 DNA Tissue kit and the EZ1 apparatus (Qiagen, Courtaboeuf, France) for further PCR-sequencing-based investigations. Ticks were identified as O. sonrai (a species not registered as an endangered species) by 16S rRNA gene sequencing, as previously described [20] (link). The ticks were tested for the presence of B. crocidurae by glpQ gene real-time PCR using a Ct≤35 cut-off [21] (link), and 18/50 (36%) ticks were found to be infected.

All collected organs were stored in Eppendorf tubes at 4°C for one day. One piece of each organ was then transferred to another 1.5 mL-Eppendorf tube containing 500 μL of sterile PBS. The organs were vigorously crushed using a single use sterile piston and 200 μL of organ juice were put into a 1.5 mL-Eppendorf tube containing a mixture of 200 μL G2 lysis buffer, 20 μL proteinase K and a small quantity of glass powder. The tube underwent three cycles of FastPrep 24^TM-5 (MP Biomedicals, Strasbourg, France) before being heated to 56°C for two hours. 200 μL of supernatant was then used to extract DNA using the EZ1 apparatus according to the manufacturer’s recommendations (Qiagen, GmbH, Germany). Extracted DNA was stored at 4°C. Detection of M. ulcerans DNA was performed by using real-time PCR (RT-PCR) and a CFX thermal cycler (BIO-Rad, Marnes-la-Coquette, France) using specific primers targeting the ketoreductase B gene (KR-b) and the IS2404 and IS2606 insertion sequences, as previously described [14 (link)]. The negative controls of our RT-PCR reactions were formed from the same reaction mix as our samples, switching only the 5μL of DNA for 5 μL of ultrapure™ DNase/RNase-Free Distilled Water (Invitrogen France). One negative control was placed after every five samples on a Light Cycler 480 multiwell plates 96-well plate (Roche).

To ensure the accuracy of the results, all precautions were taken to prevent contamination by modern louse DNA. Each experimental procedure was performed in a separate, clean room, free of P. humanus and its DNA using laminar-flow hoods, using autoclaved and UV treated material. Each louse sample was washed twice in 99.8% ethanol for 10 min, rinsed three times with distilled water and dried on sterile filter paper, and then crushed individually in sterile Eppendorf tubes. A pre-lysis of louse sample was performed in 200 μl of buffer G2 and 10 μl Proteinase K supplied in the Qiagen DNeasy tissue kit (Qiagen, Courtaboeuf, France). DNA extraction was automatically performed in the EZ1 apparatus (Qiagen, Courtaboeuf, France) using the DNeasy tissue extraction kit according to the manufacturer’s instructions. The DNA was eluted in 100 μl of in water and stored at 4 °C until use for PCR amplifications. In order to detect possible contamination by external DNA, extractions and PCR amplification blanks were used as negative controls throughout the whole process.