Spotted fever group rickettsiae in questing ticks, central Spain.

To the Editor: The number of spotted fever group (SFG) rickettsiae that cause diseases in humans is rapidly increasing (,); infections have been described in ticks and humans in Spain (,). However, in Castilla-La Mancha, central Spain, where recreational parks and hunting estates are abundant and humans may be exposed to infected ticks, information on such infections is not available. Therefore, it is worthwhile to characterize Rickettsia spp. found in this area for epidemiologic studies and proper diagnosis of possible rickettsial diseases.

In this study, we obtained 148 questing adult ticks, representing the most abundant species in the area: 12 Dermacentor marginatus, 26 Rhipicephalus bursa, 41 Rh. sanguineus, 15 Rh. turanicus, 8 Rh. pusillus, 2 Haemaphysalis punctata, 11 Hyalomma lusitanicum, and 33 Hyalomma marginatum (). The ticks were collected from the vegetation at natural sites surveyed in Castilla-La Mancha by blanket dragging with a cotton flannelette during fall 2009 and spring–summer 2010 (Figure, panel A) and classified ().

Figure

Rickettsia species in questing ticks collected in central Spain. A) Study area with 20 collection sites where ticks were found (black dots) of the 39 sites surveyed (white and black dots). B) Multilocus sequence analysis of Rickettsia spp. The evolutionary history was inferred by using the neighbor-joining method of ompA-ompB concatenated sequences (total length = 1,189 nt). The optimal tree with the sum of branch length = 0.15227017 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 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 phylogenetic relationship. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Codon positions included were 1st+2nd+3rd+noncoding. All ambiguous positions were removed for each sequence pair. Evolutionary analyses were conducted in MEGA5 (www.megasofware.net). The number of the Rickettsia spp. recognized in this study is shown next to the tick spp. Identified with them. Clusters of identified Rickettsia spp. are shown. Rc, Rickettsia conorii strain Malish 7; Ra, R. africae strain ESF-5; Rr, R. rickettsii strain Iowa; Rs, R. slovaca strain 13-B; Rm, R. massiliae strain MTU5; Rsm, R. sibirica subsp. mongolitimonae strain HA-91; R. raoultii isolate XG86; Rhsang, Rhipicephalus sanguineus; Rtur, Rh. turanicus; Rpus, Rh. pusillus; Dmar, Dermacenter marginatus; Hmar, Hyalomma marginatum. Scale bar indicates number of nucleotide changes per site.

Total DNA was extracted from dissected tick internal organs by using the DNeasy Blood & Tissue Kit (QIAGEN, Düsseldorf, Germany) and used to analyze Rickettsia spp. DNA by PCR, cloning, and sequence analysis of the amplicons. At least 3 clones were sequenced for each amplicon. Genes targeted by PCR included fragments of adenosine triphosphate synthase α subunit (atpA), heat-shock protein 70 (dnaK), outer membrane protein A (ompA), outer membrane protein B (ompB), citrate synthase (gltA), 16S rRNA, recA, and initiator protein of DNA replication (dnaA) (,). To characterize Rickettsia spp., we compared nucleotide sequence identity to reference strains and carried out multilocus analysis using ompA-ompB sequences and in silico PstI and RsaI restriction analysis of ompA sequences ().

Ticks were first screened by 16S rRNA PCR, and positive samples were analyzed for all targeted genes. The results showed that 27 (18.2%) of the 148 ticks analyzed were positive for Rickettsia spp. Of these, 11 were confirmed as R. massiliae in Rh. sanguineus, Rh. turanicus, and Rh. pusillus, 3 as R. raoultii in D. marginatus, 2 as R. slovaca in D. marginatus, and 2 as R. sibirica subsp. mongolitimonae in H. marginatum and Rh. pusillus (Figure, panel B). These species had >99% pairwise nucleotide sequence identity to reference strains R. massiliae MTU5 (GenBank accession no. NC_009900), R. slovaca 13-B (accession no. NC_016639), and R. sibirica subsp. mongolitimonae HA-91 (accession no. AHZB00000000) genome sequences for all genes analyzed, and the only R. raoultii reported sequences (accession nos. JQ792107, JQ792166, JQ792134, and NR_043755 for ompB, ompA, gltA, and 16S rRNA, respectively). The sequences obtained in this study were deposited in the GenBank under accession nos. KC427998–KC428040.

Multilocus sequence analysis of ompA-ompB sequences (Figure, panel B) and in silico PstI and RsaI restriction analysis of ompA sequences also confirmed the identity of the Rickettsia spp. identified in this study. As previously shown (,), multilocus analysis with ompA-ompB sequences was highly informative about the phylogenetic relationship between Rickettsia spp. (Figure, panel B), with similar results for maximum likelihood, maximum parsimony, and neighbor-joining methods (data not shown). Furthermore, the results suggested the tick vectors for these Rickettsia spp. in the study area (Figure, panel B) match those reported or suspected previously for these Rickettsia spp. (–), but for the first time, R. sibirica subsp. mongolitimonae was identified in Hyalomma and Rhipicephalus spp. ticks in Spain ().

These tick species are frequently found in the same area feeding on Eurasian wild boar (Sus scrofa) and red deer (Cervus elaphus), which may act as hosts for these pathogens (,). To test this hypothesis, we determined the seroprevalence for SFG rickettsiae in these host species in Castilla-La Mancha. Serum samples from 235 red deer and 206 wild boar were analyzed for the presence of anti-SFG Rickettsia antibodies by ELISA (Spotted Fever Rickettsia IgG EIA Antibody Kit, Fuller Laboratories, Fullerton, CA, USA). The ELISA was adapted to test ungulate serum specimens by substituting antihuman IgG-horseradish by protein G-horseradish peroxidase (Sigma-Aldrich, Madrid, Spain). Specific SFG-Rickettsia antibodies were detected in 146 (70.9%) of 206 wild boar and 174 (74.0%) of 235 red deer, indicating a high seroprevalence in these species and thus the possibility that they can serve as hosts for these pathogens.

These tick species also infest humans, thus posing a risk for transmission of rickettsiae that are pathogenic in humans (). In fact, Castilla-La Mancha is one of the regions in Spain where a high number of SFG rickettsioses are reported ([]; http://pagina.jccm.es/sanidad/salud/epidemiologia/3507.pdf).

In conclusion, these results demonstrate that SFG rickettsiae with public health relevance are found in ticks in central Spain as in other regions in Spain. In central Spain, the widespread distribution of tick vectors and possible wildlife hosts, the presence of persons in tick-infested recreational and hunting areas, and the transstadial and transovarial transmission of the pathogen in ticks may favor transmission to humans.