CP001612_R. africae, AH015610_R. raoultii, AY319290_R.
rickettsia, KF702333_Candidatus Rickettsia, KY780025_R. sibirica,
EU272186_ Candidatus Rickettsia, MF511254_R. raoultii, MH500082 _R.
mongolotimonae, MH532255_R.slovaca, KY113110_R.parkeri,
KY233245_CANDIDATUS Rickettsia barbariae, MF379309_R. sibirica,
KY513920_R. sibirica, EU622980_R.africae, MH532254_R.slovaca,
MG515014_R. africae GU247115_R. africae, KJ645933_R. africae,
U43790_R. africae, DQ103259_R. tamurae, LC388791_R.
tamurae,KC003476_R. parkeri,KU744412_R. parkeri
Figure 1: Neighbour-joining rectangular cladogram of 12S mitochondrial
rDNA of tick species generated from the study with the reference
sequences from GenBank. The cladogram was constructed by using the
neighbour-joining method in ClustalX 2.1 program. Test sequences
intermingled with sequences of Amblyomma, Hyalomma, andRhipicephalus species reference strains obtained from GenBank.
All sequenced ticks samples clustered with the three identified genera;
Amblyomma, Hyalomma and Ripicephalus reference sequences obtained from
GenBank. Test sequences are in bracket and arrowed while the reference
sequences are in GenBank accession number.
Figure 2a: Nucleotide sequence alignment of the ompB gene of
sample B188 against the homologous reference sequence of R.
parkeri (KY113111) indicating 100% homology. Reference sequence was
obtained based on highest percentage homology that the test sequence has
with R. parkeri which was obtained through Nucleotide BLAST tool
in the GenBank. The dots represent nucleotide similarity of the query
sequence with the reference strain.
Figure 2b: Amino acid sequence alignment of the ompB gene of
sample B188 against the homologous reference sequence of R.
parkeri (KY113111) indicating 100% homology. Reference sequence was
obtained based on highest percentage homology that the test sequence has
with R. parkeri which was obtained through Nucleotide BLAST tool
in the GenBank. The dots represent amino acid similarity of the query
sequence with the reference strain.
Figure 2c: Nucleotide sequence alignment of the ompA gene of
sample A188 against the homologous reference sequence ofRickettsia sp. strain Ga-Seema (MG953288) indicating
95% homology. Reference sequence was obtained based on highest
percentage homology that the test sequence has with Rickettsiasp. strain Ga-Seema which was obtained through Nucleotide BLAST tool in
the GenBank. The dots represent nucleotide similarity of the query
sequence with the reference strain.
Figure 2d: Amino acid alignment of the ompA gene of sample A188
against the homologous reference sequence of Rickettsia sp.
strain Ga-Seema (MG953288) indicating 95% homology. Reference
sequence was obtained based on highest percentage homology that the test
sequence has with Rickettsia sp. strain Ga-Seema which was
obtained through Nucleotide BLAST tool in the GenBank. The dots
represent amino acid similarity of the query sequence with the reference
strain.
Figure 3a: Nucleotide sequence alignment of the ompB gene of
sample B209 against the homologous reference sequence of R.
tamurae (DQ113910) indicating 100% homology. Reference sequence was
obtained based on highest percentage homology that the test sequence has
with R. timurae which was obtained through Nucleotide BLAST tool
in the GenBank. The dots represent nucleotide similarity of the query
sequence with the reference strain.
Figure 3b: Amino acid sequence alignment of the ompB gene of
sample B209 against the homologous reference sequence of R.
tamurae (DQ113910) indicating 100% homology. Reference sequence was
obtained based on highest percentage homology that the test sequence has
with R. timurae which was obtained through Nucleotide BLAST tool
in the GenBank. The dots represent amino acid similarity of the query
sequence with the reference strain.
Figure 3c: Nucleotide sequence alignment of the ompA gene
of sample A209 against the homologous reference sequences of R.
tamurae (DQ103259) and R. africae (EU622980) indicating 100%
identity with R. africae whereas the ompB gene of the same
sample has 100% identity with R. tamurae. Reference sequence
was obtained based on highest percentage homology that the test sequence
has with R. africae which was obtained through Nucleotide BLAST
tool in the GenBank. The three nucleotides insert that differentiateR. africae and R. tamurae are underlined at position 97 to
99. The dots represent nucleotide similarity of the query sequence with
the reference strain.
Figure 3d: Amino acid sequence alignment of the ompA gene
of sample A209 against the homologous reference sequences of R.
tamurae (DQ103259) and R. africae (EU622980) indicating 100%
identity with R. africae whereas the ompB gene of the same
sample has 100% identity with R. tamurae. Reference sequence
was obtained based on highest percentage homology that the test sequence
has with R. africae which was obtained through Nucleotide BLAST
tool in the GenBank. The three nucleotides insert that differentiateR. africae and R. tamurae are underlined at position 97 to
99. The dots represent amino acid similarity of the query sequence with
the reference strain.
Figure 4: Phylogenetic tree of ompA gene sequences in
bold generated from the study with the related reference sequences
obtained from NCBI GenBank. The evolutionary history was inferred using
the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree
with the sum of branch length = 1.67432413 is shown. The percentage of
replicate trees in which the associated taxa clustered together in the
bootstrap test (1000 replicates) is shown next to the branches
(Felsenstein, 1985). The evolutionary distances were computed using the
p-distance method (Nei and Kumar, 2000) and are in the units of the
number of base differences per site. The analysis involved 67 nucleotide
sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All
positions containing gaps and missing data were eliminated. There were a
total of 166 positions in the final dataset. Evolutionary analyses were
conducted in MEGA7 (Kumar et al., 2016). The test sequences denoted
diamond red clustered with other Rickettsia references.
Figure 5: Phylogenetic tree
of ompB gene sequences in bold generated from the study with the
related reference sequences obtained from NCBI GenBank. The
evolutionary history was inferred using the Neighbor-Joining method
(Saitou and Nei, 1987). The optimal tree with the sum of branch length =
2.89828155 is shown. The percentage of replicate trees in which the
associated taxa clustered together in the bootstrap test (1000
replicates) is shown next to the branches (Felsenstein, 1985). The
evolutionary distances were computed using the p-distance method18 and are in the units of the number of base
differences per site. The analysis involved 86 nucleotide sequences.
Codon positions included were 1st+2nd+3rd+Noncoding. All positions
containing gaps and missing data were eliminated. There were a total of
236 positions in the final dataset. Evolutionary analyses were conducted
in MEGA7 (Nei and Kumar, 2000). All study sequences clustered
phylogenetically with R. africae sequences from GenBank with the
exception of sequences B188 and B209; B188 clustered with R.
parkeri (AF123717) while sample B209 clustered with R. tamurae(DQ113910) with high bootstrap values above 99%. Test sequences are in
bold in green dot.