Molecular characterization of Borrelia persica, the agent of tick borne relapsing fever in Israel and the Palestinian Authority

Abstract

The identification of the Tick Borne Relapsing Fever (TBRF) agent in Israel and the Palestinian Authority relies on the morphology and the association of Borrelia persica with its vector Ornithodoros tholozani. Molecular based data on B. persica are very scarce as the organism is still non-cultivable. In this study, we were able to sequence three complete 16S rRNA genes, 12 partial flaB genes, 18 partial glpQ genes, 16 rrs-ileT intergenic spacers (IGS) from nine ticks and ten human blood samples originating from the West Bank and Israel. In one sample we sequenced 7231 contiguous base pairs that covered completely the region from the 5'end of the 16S rRNA gene to the 5'end of the 23S rRNA gene comprising the whole 16S rRNA (rrs), and the following genes: Ala tRNA (alaT), Ile tRNA (ileT), adenylosuccinate lyase (purB), adenylosuccinate synthetase (purA), methylpurine-DNA glycosylase (mag), hypoxanthine-guanine phosphoribosyltransferase (hpt), an hydrolase (HAD superfamily) and a 135 bp 5' fragment of the 23S rRNA (rrlA) genes. Phylogenic sequence analysis defined all the Borrelia isolates from O. tholozani and from human TBRF cases in Israel and the West Bank as B. persica that clustered between the African and the New World TBRF species. Gene organization of the intergenic spacer between the 16S rRNA and the 23S rRNA was similar to that of other TBRF Borrelia species and different from the Lyme disease Borrelia species. Variants of B. persica were found among the different genes of the different isolates even in the same sampling area.

Figure 1. Physical map of the rrs-rrlA genomic region (7231 contig) of Borrelia persica.

The genes and their orientation are indicated by empty arrows. The position of each locus on the 7231 contig is given in parentheses. Positions of nucleotides polymorphism are indicated by black arrows. Nucleotide changes resulting in amino acid modifications are marked with an asterisk.

Figure 2. Phylogenetic tree based on the rrs-rrlA spacer region.

The tree was inferred using the UPGMA method. The bootstrap consensus tree inferred from 250 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (250 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 7106 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (9).

Figure 3. Phylogenic tree based on rrs-ileT spacer (IGS) sequences.

The rrs-ileT spacer (IGS) sequences for 16 independent isolates from Israel and the West Bank belonging to genovars a to c were compared to IGS sequences from other Borrelia species (accession numbers are given in parentheses). The isolates in each genovar are listed in Table 1. The phylogenic tree was inferred using the UPGMA method as described in Figure 2. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 349 positions in the final dataset.

Figure 4. Phylogenic tree based on rrs sequences.

The complete rrs sequences of B. persica isolates in Israel were compared to rrs sequences from B. persica (Iran) and other Borrelia species (accession numbers are given in parentheses). The phylogenic tree was inferred using the UPGMA method. Parameters were as described in Figure 2. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 1522 positions in the final dataset.

Figure 5. Phylogenetic tree based on purA nucleotide sequences.

The complete purA sequences of B. persica isolates in Israel and the West Bank were compared to purA sequences from other Borrelia species (accession number are given in parentheses). The Phylogenic tree was inferred using the UPGMA method. Parameters were as described in Figure 2. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 1284 positions in the final dataset.

Figure 6. Phylogenic tree based on glpQ nucleotide sequences.

glpQ sequences of 18 independent isolates from Israel and the West Bank belonging to genovars G1 to G4 were compared to glpQ sequences from other Borrelia species (accession numbers are given in parentheses). The isolates in each genovar are listed in Table 1. The phylogenic tree was inferred using the UPGMA method as described in Figure 2. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 637 positions in the final dataset.

Figure 7. Phylogenetic tree based on the concatenated alignments of purA, glpQ, flaB and rrs-ileT IGS nucleotide sequences.

The tree was inferred using the PHYML program. The percentage of trees in which the associated taxa clustered together in the bootstrap test (100 replicates) is shown next to the branches if higher than 80%. The tree was arbitrarily rooted at midpoint.