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.