Molecular Characterization of Borrelia persica, the Agent of Tick Borne Relapsing Fever in Israel and the Palestinian Authority

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

Phylogenetic tree based on <i>pur</i>A nucleotide sequences.

<p>The complete <i>pur</i>A sequences of <i>B. persica</i> isolates in Israel and the West Bank were compared to <i>pur</i>A sequences from other <i>Borrelia</i> species (accession number are given in parentheses). The Phylogenic tree was inferred using the UPGMA method. Parameters were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014105#pone-0014105-g002" target="_blank">Figure 2</a>. 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.</p

Phylogenic tree based on <i>rrs-ile</i>T spacer (IGS) sequences.

<p>The <i>rrs-ile</i>T 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 <i>Borrelia</i> species (accession numbers are given in parentheses). The isolates in each genovar are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014105#pone-0014105-t001" target="_blank">Table 1</a>. The phylogenic tree was inferred using the UPGMA method as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014105#pone-0014105-g002" target="_blank">Figure 2</a>. 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.</p

Variable nucleotide positions and genovar definition based on the <i>pur</i>A gene of 8 human and tickborne isolates of <i>B. persica</i> in Israel and the West Bank.

<p>nd: not done.</p>1<p>List of isolates in each genovar is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014105#pone-0014105-t001" target="_blank">Table 1</a>.</p>2<p>Positions of nucleotide changes are numbered according to the 7231 contig (HM131216) (this work). Where relevant, the resulting amino acid modification is shown in parenthesis.</p

List of the primers used in this work.

1<p>An s in a primer's name indicates that it was used for sequencing only. Primers marked with n were used in nested PCR reactions.</p

Phylogenic tree based on <i>glp</i>Q nucleotide sequences.

<p><i>glp</i>Q sequences of 18 independent isolates from Israel and the West Bank belonging to genovars G1 to G4 were compared to <i>glp</i>Q sequences from other <i>Borrelia</i> species (accession numbers are given in parentheses). The isolates in each genovar are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014105#pone-0014105-t001" target="_blank">Table 1</a>. The phylogenic tree was inferred using the UPGMA method as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014105#pone-0014105-g002" target="_blank">Figure 2</a>. 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.</p

Characterization of the isolates investigated in this work.

1<p>Typing and genovar determination were performed for the genes encoding Flagellin (<i>fla</i>B), Glycerophosphodiester phosphodiesterase (<i>glp</i>Q), 16S rRNA (rrs), Adenylosuccinate synthetase (<i>pur</i>A), and the intergenic spacer (IGS) between <i>rrs</i> and the ile tRNA (<i>ile</i>T) and between <i>rrs</i> and the 23S rRNA (<i>rrlA</i>).</p>2<p>nd: not done.</p

Coefficient of similarity between genetic loci of local <i>B. persica</i> and other TBRF <i>Borrelia</i> species.

<p>na: not available.</p><p>All local strains were included in the assessment of similarity range among <i>B. persica</i> isolates studied in this work. We used the sequences of <i>B. persica</i> strain HL2610 (relevant accession numbers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014105#s2" target="_blank">materials and methods</a>) for comparison with other TBRF <i>Borrelia</i> available homologous genes: <i>B. persica</i> Iran <i>rrs</i> (U42297), <i>glp</i>Q of <i>B. persica</i> Iran (EU914143), <i>B. hispanica rrs</i> (U42294), <i>B. hispanica rrs-ileT</i> IGS (FJ827590), <i>B. recurrentis</i> complete genome (CP000993), <i>B. duttonii</i> complete genome (CP000976), <i>B. hermsii</i> complete genome (CP000048) and <i>B. turicatae</i> complete genome (CP000049). Vector NTI advance 11 software was used for sequence alignments.</p