Detection of bacterial and protozoan pathogens
Tick, louse, flea, and livestock-blood samples were also screened for bacteria and protozoa using a combination of PCR-HRM and conventional PCR. Previously developed primers that target the 16S rRNA gene ofAnaplasma (Mwamuye et al., 2017), Ehrlichia (Mwamuye et al., 2017), and Rickettsia (Nijhof et al., 2007), as well as primers that target the 18S ribosomal gene of Theileria andBabesia parasites (Georges et al., 2001), were used for initial screening (Supplementary Table 1). Ten-microliter reactions that consisted of 2 µl template, 2 µl 5X HOT FIREPol® EvaGreen HRM Mix (Solis BioDyne, Estonia), and 0.5 µl of each primer at 10 µM concentrations. Cycling was carried out in a Rotor-Gene Q real-time PCR thermocycler (Qiagen, Hilden, Germany) as described before (Mwamuye et al., 2017). Positive controls for Anaplasma (A. marginale ) andRickettsia (R. africae ) (previously detected inicipe ’s ML-EID lab from Amblyomma spp. ticks) were included in the runs. Resultant HRM profiles were visually inspected with Rotor-Gene Q Series software 2.1.0 and representative amplicons with unique HRM profiles were purified using an Exo 1-rSAP combination (Biolabs, UK) and sequenced at Macrogen (Netherlands).
Positive Ehrlichia and Anaplasma samples were further amplified with a semi-nested PCR to generate a longer fragment of the 16S rRNA gene (1030 bp) by combining the Anaplasmataceae-specific forward primer, EHR16SD (Parola et al., 2001) with universal reverse primers pH1522 (Edwards et al., 1989) and pH1492 (Reysenbach et al., 1992) for first and second round amplification, respectively (Supplementary Table 1). Primary amplifications were performed using a hot-start activation step of 95°C for 15 min followed by 1 cycle of 95°C for 20 s, 63°C for 30 s, and 72°C for 90 s, 2 cycles of 95°C for 20 s, 62°C for 30 s, and 72°C for 90 s, 2 cycles of 95°C for 20 s, 61°C for 30 s, and 72°C for 90 s, followed with 35 cycles of 95°C for 20 s, 60°C for 30 s, and 72°C for 80 s, and a final extension at 72°C for 10 min. The secondary 20-µl amplification reactions utilised 2 µl of PCR products from primary reactions as templates. The cycling profile consisted of: 95°C for 15 min; 3 cycles of 95°C for 20 s, 61°C for 30 s, and 72°C for 90 s; 37 cycles of 95°C for 20 s, 60°C for 30 s, and 72°C for 80 s, and a final extension at 72°C for 10 min. To minimise the risk of contamination we set up the second reaction in a PCR enclosure and opened only one tube at a time. Products were visualised after gel electrophoresis to confirm the presence of the expected product at 1030bp. For Rickettsia , all samples with positive HRM profiles were further amplified with Rick-ompB primers (Roux & Raoult, 2000) targeting a 856-bp region of the outer membrane protein B gene of all Rickettsia species (Supplementary Table 1). Positive samples were prepared for sequencing using the QuickClean II Gel Extraction Kit (GenScript, New Jersey, USA) and submitted to Macrogen (Netherlands) for bidirectional sequencing.