Detection, identification and prevalence ofRickettsia spp. in the study sites

A total of 60 (6.3%) positive samples for the three genes amplified were detected for Rickettsia spp. from the 953 ticks analyzed. PCR assays used for the identification of Rickettsia spp. were positive for all genes fragments profiled per every positive sample of which only some ompA (43) and all of omp B (60) amplicons were sequenced and analyzed to delineate the Rickettsia spp. down to the species level.
Rickettsial DNA fragments were obtained from the three genera of ticks collected in the study sites with more detected in A. hebreaum.Also, a higher proportion of the pathogens (55%) were obtained in ticks collected from cattle, 23% prevalence in ticks from goats while that of sheep was 22%.
A homology search for generated Rickettsia sequences showed that they had a high sequence similarity of above 97% with homologous sequences of Rickettsia in GenBank. Comparison of the 43omp A and 60 omp B sequences by BLAST analysis showed that majority of the sequences have ≥98% homology with R. africaewhile only two samples (B188) and B209 had 99% similarity with R. parkeri (KY113111) and 100% homologous with R. tamurae(DQ113910) respectively based on omp B BLASTn analysis.However, discordances in homology between omp A and omp B sequences of the two samples were observed as shown in Figures 2a-d and 3a-d respectively. The omp A of sample 188 showed 99% similarity with Candidatus Rickettsia (EU272186) while the omp B of the same sample was 100% homologous to R.parkeri (KY113111) in both amino acid and nucleotide sequences alignments. Similarly, A209 of sample 209 was 100% homologous with R. africae (EU622980) while B209 of the same sample was 100% homologous with R.tamurae(DQ113910) respectively in both amino acid and nucleotide sequences alignments. Nucleotides and amino acids sequences alignments were performed with the corresponding homologous sequences of omp A andomp B sequences as shown in Figures 2A to D and 3A to D respectively.
Reference sequences used for phylogenetic analysis were randomly selected and analyzed with generated study sequences of omp A andomp B genes. The phylogenetic tree obtained for ompA gene showed that test sequences clustered with reference sequences from NCBI GenBank nucleotides database as illustrated in Figure 4. Majority of theomp A sequences clustered with R. africae (U43790; KJ645933, GU247115, MG515014) with bootstrap values above 81% while sequences A196 and A198 clustered equidistance between R.tamurae(LC388791) R.africae (CP001612) with 92% bootstrap reliability and A188 and A208 clustered with Candidatus Rickettsia EU272186 with 77% bootstrap value as shown in Figure 4 while the phylogenetic tree constructed with omp B sequences is shown in Figure 5 with bootstrap values above 70%. Phylogenetic analyses of the omp A and omp B sequences support the observed discrepancies in homology. Sequence B150 clustered between R.rhipicephali(AF123719) and R.parkeri (KY113111) phylogenetically but a BLAST search showed it as having close homology with UnculturedRickettsia clone and R. conorii (FJ015092) by 76% respectively.

GenBank accession numbers

Sequences obtained in this study have been deposited in the GenBank database under the following accession numbers: MK347206-MK347212 (tick identification), MK405447-MK405477 (rickettsia omp A gene), MK405386-MK405446 (rickettsia omp B gene).