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.