Rickettsia felis in Rhipicephalus sanguineus from Two Distant Chilean Cities

Introduction

R ickettsia felis is a relatively new member of the spotted fever group (SFG) of the genus Rickettsia. Its worldwide presence and the wide range of ectoparasites and mammals, including humans, in which it has been found, make it a threat for human health (Pérez Osorio et al. 2008). Although its recognized vector and natural reservoir is Ctenocephalides felis, the cat flea (Galvao et al. 2006, Pérez Osorio et al. 2008, Reif and Macaluso 2009) R. felis has also been found in other flea species, some mosquitoes, and in different ticks, including Rhipicephalus sanguineus (Cardoso et al. 2006, Reif and Macaluso 2009).

In Chile, R. felis was found some years ago in 21/30 cat fleas taken from several cats from one house (Labruna et al. 2007) and in 1/100 specimens of R. sanguineus (López, unpublished data); in both cases, the ectoparasites were taken from pets living in Santiago, Metropolitan Region (MR). Whether the presence of R. felis is widespread in Chile is unknown. Therefore, the purpose of this study was determining the presence of Rickettsia spp. in R. sanguineus, the main canine tick in the country, in two different geographical areas of the country.

Materials and Methods

The study was performed in the MR, central region of Chile (33°45′S,70°76′W), and in the northern city of Arica (18°28′S, 70°18′W). A random stratified sampling of households of one municipality of MR (Puente Alto) and of Arica city were carried out, besides a convenience sampling in a rural municipality of MR (Pirque) and in rural areas near Arica city. Domiciliary visits to pet-owning households were performed during the Chilean tick season (September, 2010, to February, 2011) by a veterinary team who collected ticks from one dog per household. Tick species identification was determined by taxonomic keys.

DNA extraction from crushed R. sanguineus was performed using Wizard Genomic DNA Purification Kit (Promega, Madison, WI) and then processed by PCR targeting the conserved citrate synthase (gltA) gene using CS2-78 and CS2-323 primers and published protocols (Labruna et al. 2004). Positive samples were used for a second round of PCR targeting the outer membrane protein ompA gene of the SFG using Rr-190.70p and Rr-190.701 primers (Roux et al. 1996) and ompB protein gene using BG1-21F and BG2-20 primers. Conditions for ompA and ompB PCR were the same used for gltA with one change in the annealing temperature (48°C and 55°C, respectively). In each set of reactions, negative (water) and positive (DNA of Rickettsia parkeri strain NOD) controls were included. PCR products were separated on 1.5% agarose gel, stained with ethidium bromide, and visualized under UV light. Samples with amplicons of the expected size were purified using Wizard^® SV Gel and PCR Clean-Up System and DNA sequenced in an automatic sequencer (Macrogen, Bethesda, MD). Partial sequences obtained were compared with others in the GenBank using BLAST analysis.

An exact Fisher test was used for statistical comparisons.

Results

Overall, 460 dogs were included in the study, of them 236 (50.2%) were infested by R. sanguineus. Canine infestations rates varied from 32 to 75% among urban and rural areas of the sampled localities (Table 1).

Partial sequences of gltA and ompA genes were obtained from nine R. sanguineus ticks, from which four also yielded ompB partial sequences. Sequences of gltA were identical between them; BLAST analysis showed 99–100% identity with R. felis from California (CP000053.1). They were incorporated to GenBank under accession numbers JF751024, JX298087, JX298088, JX298089, JX298090, JX298091, and JX298092. R. felis PCR-positive ticks corresponded to dogs from urban and rural localities of Arica and from rural MR. No differences were found in frequency of positive ticks between urban and rural sites within each region, but a higher frequency was found in Arica than in MR (p=0.002).

Discussion

R. felis is an emergent pathogen described in more than 25 countries on five continents. Due to its wide range of hosts and the ability of its main vector C. felis to bite humans, this is considered a threat to human health (Pérez Osorio et al. 2008). In a medical literature search, 68 human cases were reported (Renvoise et al. 2009), including some from Mexico and Brazil (Galvao et al. 2006). Due to the nonspecificity of the clinical picture and the lack of availability of diagnostic tests, the infection seems to be vastly unrecognized (Pérez Osorio et al. 2008).

Although most of the reports have found R. felis in C. felis, it has been also found in other flea species, ticks, and mosquitoes (Cardoso et al. 2006, Reif and Macaluso 2009). In Latin America, there are reports of its presence in C. felis in Mexico, Brazil, Peru, Chile, Argentina, and Uruguay (Cardoso et al. 2006, Galvao et al. 2006, Labruna et al. 2007, Nava et al. 2008) and from R. sanguineus in Brazil and Chile (Cardoso et al. 2006, López, unpublished data). To date, the role of this tick in the ecology and transmission of R. felis has not been clarified (Reif and Macaluso 2009); its presence in ticks could be just the result of a blood meal from an animal infected by a flea.

R. sanguineus is the main tick infesting Chilean dogs. In rural areas, Amblyomma tigrinum and Amblyomma triste have been described, with the latter first reported by us in rural localities of Arica (Abarca et al. 2012). Herewith we confirm the presence of R. felis in R. sanguineus in Chile, in a wide geographical extension that include a semirural municipality of MR in Santiago and the city and rural localities of Arica in the Northern region, bordering Peru and Bolivia. If future studies confirm an ability of R. sanguineus to transmit R. felis, our results would highlight the need of both tick and flea control. Considering the evidence of human infections associated with R. felis (Galvao et al. 2006, Renvoise et al. 2009), our results highlight the relevance of the endemic presence of this agent in Chile, where to date no cases of human SFG cases have been recognized.