Table A.

Genes and primers used in polymerase chain reaction (PCR) assays to detect Coxiella and relatives and to control tick DNA quality. The same primers were used for the Coxiella and Rickettsiella typing, with the exception of the 16S rRNA gene for which different primers were designed for the two bacteria. Nested PCR amplifications (16S rRNA, 23S rRNA, GroEL, rpoB and dnaK) were performed as follows: the first PCR run with the external primers was performed in a 10μLvolume containing 20–50 ng of genomic DNA, 3 mM of each dNTP (Thermo Scientific), 8 mM of MgCl2 (Roche Diagnostics), 3 μM of each primer, 1 μL of 10× PCR buffer (Roche Diagnostics), and 0.5 U of Taq DNA polymerase (Roche Diagnostics). A 1-μL aliquot of the PCR product from the first reaction was then used as a template for the second round of amplification. The second PCR was performed in a total volume of 25 μL and contained 8 mM of each dNTP (Thermo Scientific), 10 mM of MgCl2 (ThermoScientific), 7.5 μM of each of the internal primers, 2.5 μL of 10×PCR buffer (Thermo Scientific), and 1.25 U of Taq DNA polymerase (Thermo Scientific). Non-nested PCR amplifications (CO1 and 18S rRNA) were performed following conditions similar to the first PCR run used in the nested PCR assays. All PCR amplifications were performed under the following conditions: initial denaturation at 93°C for 3 min, 35 cycles of denaturation (93°C, 30 s), annealing (Tm = 50–56°C, depending on primers, 30 s), extension (72°C, 1–2 min), and a final extension at 72°C for 5 min. Table B. List, biological features and GenBank accession numbers of the bacterial strains used as references in molecular and phylogenetic analyses.* reference strains of C. burnetii used for primer testing. Table C. List, sequence accession numbers and features of the 31 Coxiella contigs from the whole-genome shotgun sequencing (WGS) of the cattle tick Rhipicephalus microplus. Fig A. Map of the Coxiella burnetii genome (strain Nine Mile I RSA 493) showing the position of the genetic markers (in blue) used in this study. The arrows indicate the position along the chromosome of the five housekeeping genes (16S rRNA, 23S rRNA, GroEL, rpoB and dnaK) used in the multi-locus typing of tick-borne Coxiella infections. The numbered boxes (1–31) indicate the position of the 31 Coxiella contigs (listed in Table C in S1 Text) detected from the whole genome sequencing of the hard tick Rhipicephalus microplus. Fig B. Coxiella and Rickettsiella phylogeny constructed using maximum-likelihood (ML) estimations based on 16S rRNA, 23S rRNA, GroEL, rpoB and dnaK concatenated sequences (3009bp), including 71 Coxiella-like strains of ticks, 15 C. burnetii reference strains and outgroups. The four Coxiella clades are labeled A to D. Each number corresponds to one tick species as detailed in Table 1. Blue, Coxiella-line organisms; red, C. burnetii; green, Rickettsiella; black, other bacteria. All multi-locus typing of tick-borne Coxiella and Rickettsiella of ticks are new sequences from this study. Branch numbers indicate percentage bootstrap support for major branches (1000 replicates; only bootstrap values >90% are shown). Fig C. Inset of Coxiella network from Fig 2 with focus on the A clade (Coxiella of soft ticks and C. burnetii). Each number corresponds to one tick species as detailed in Table 1. Blue, Coxiella-line organisms; red, C. burnetii. The scale bar is in units of substitution/site.
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