Total genomic DNA extraction from ticks
Prior to DNA extraction, the preserved specimens were washed with
sterile distilled water and left to dry in a sterile Petri dish. This
was done in order to remove all the ethanol residues from the tick
samples which might negatively affect the downstream PCR reactions. The
ticks were chopped into bits with sterile blades in the Petri dishes
based on their morphological delineations and processed separately for
DNA extraction using ReliaPrep DNA Tissue miniprep system ZYMORESEARCH
Quick DNA Universal Kit according to the manufacturer’s instruction and
as previously described by Iweriebor et al. (2017). The adult engorged
and non-engorged ticks were processed separately while the nymphs of the
same species collected from the same animal were pooled together and
processed for DNA extraction. All ticks processing and PCR reactions
were performed in biosafety cabinet which were always left sterile
overnight using UV light. Absolute precautionary measures were adopted
to avoid cross contaminations of reagents, blades and instruments used
throughout the process as well as pipettor sets were dedicated to
specific functions, i.e., reagent dispensing, template isolation, PCR
setup, and template handling. Commercially certified DNA/RNase free
filter barrier tips were used to prevent aerosol contamination. PCR
setup was performed far from the isolation materials, and reagent
handling was separated both physically and temporally from templates.
Molecular identification of tick and detection of
bacteria in ticks
Each tick specimen was assessed using PCR for both tick identification
and detection of Rickettsia spp. Confirmation of tick species
identification was done by PCR targeting the mitochondrial 12S rDNA gene
from representatives of the genera hitherto identified morphologically
(Yawa et al., 2016; Williamson et al., 2010). Screening forRickettsia spp. was performed using three of the primer pairs as
indicated in Table 1 that are used for the species identification ofRickettsia pathogens as previously reported by Regnery et al.
(1991) and Kollars and Kengluecha. (2001) in a PCR assay as adapted by
Williamson et al., (2010)
Table 1. Primer sequences used in
the molecular identification of ticks and Rickettsia spp
Primer name Gene Primer Sequence (5’ to3’) Amplicon bp TM Ref
Tick DNA
85F 12S TTAAGCTTTTCAGAGGAATTTGCTC 110 54.0 Walker et al., 2003
225R 12S TTTWWGCTGCACCTTGACTTAA 52.7
Rickettsia spp.
Rr.190 70P rompA ATGGCGAATATTTCTCCAAAA 610 52.5 Regnery et al.,
1991
Rr.190 602N rompA AGTGCAGCATTCGCTCCCCCT 64.9
BG1-21 rompB GGCAATTAATATCGCTGACGG 511 55.6 Eremeeva &Raoult,
1994
BG2-20 rompB GCATCTGCACTAGCACTTTC 55.2
RrCS372 gltA TTTGTAGCTCTTCTCATCCTATGGC 410 59.0 Kollars and
Kengluecha,2001
RrCS989 gltA CCAAGTTCCTTTAATACTTCTTTGC 57.4
DNA sequencing, sequence editing and BLASTn
search
The amplified PCR products were sequenced in a commercial sequencing
facility. Only 43 samples out of the 60 positive omp A amplicons
were sequenced due to some technical constraints and all the positiveomp B amplicons were sequenced while all the amplified
representatives of tick’s 12S rDNA amplicons were also sequenced and
none was sequenced for the glt A positive amplicons. The inability
to sequence the glt A amplicons was due to financial constraint
coupled with the fact that the discriminatory power to delineateRickettsia spp. by the three amplified genes are equal and as
such any of the genes could suffice for delineation of the species among
the genus Rickettsia . The alignment and sequence editing was
achieved using Geneious Prime 2019.0.3 version. The generated nucleotide
sequences were subjected to homology search using the Basic Local
Alignment Search Tool (National Center for Biotechnology Information,
www.ncbi.nlm.nih.gov) where the
edited sequences were compared with representative genetic sequences of
previously characterized rickettsial and tick species curated in the
GenBank NCBI. Rickettsial sequences that had above 97% homology with
test samples were used as reference strains for phylogenetic analyses
along with other curated representatives of SFGR sequences in the
GenBank.
3.1.7 Phylogenetic analysis
Edited sequences were aligned with reference sequences obtained from
GenBank after the BLAST search which was used to construct a maximum
possible phylogenetic tree using Mega 7 version software with 1000
bootstrap replicate. Phylogenetic trees were constructed using BioEdit
Tree Builder. Sequence data sets for positive samples were submitted to
NCBI GenBank for accession numbers.