Rickettsia species infecting Amblyomma ticks from an area endemic for Brazilian spotted fever in Brazil

This study reports rickettsial infection in Amblyomma cajennense and Amblyomma dubitatum ticks collected in an area of the state of Minas Gerais, Brazil, where Brazilian spotted fever is considered endemic. For this purpose, 400 adults of A. cajenennse and 200 adults of A. dubitatum, plus 2,000 larvae and 2,000 nymphs of Amblyomma spp. were collected from horses and from the vegetation. The ticks were tested for rickettsial infection through polymerase chain reaction (PCR) protocols targeting portions of three rickettsial genes (gltA, ompA, and ompB). Only two free-living A. cajennense adult ticks, and four pools of free-living Amblyomma spp. nymphs were shown to contain rickettsial DNA. PCR products from the two A. cajennense adult ticks were shown to be identical to corresponding sequences of the Rickettsia rickettsii strain Sheila Smith. DNA sequences of gltA-PCR products of the four nymph pools of Amblyomma spp. revealed a new genotype, which was shown to be closest (99.4%) to the corresponding sequence of Rickettsia tamurae. Our findings of two R. rickettsii-infected A. cajennense ticks corroborate the endemic status of the study area, where human cases of BSF were reported recently. In addition, we report for the first time a new Rickettsia genotype in Brazil.


Introduction
The ixodid ticks Amblyomma cajennense and Amblyomma dubitatum are the most frequent ectoparasites of capybaras (Hydrochoerus hydrochaeris) in Southeastern Brazil (VIEIRA et al., 2004;PACHECO et al., 2007).In addition, A. cajennense is also a common parasite of horses, dogs, and various medium to large-sized wild mammals, and is the most common biting tick affecting humans in Southeastern and Central-Western Brazil as well (VIEIRA et al., 2004;GUGLIELMONE et al., 2006).On the other hand, A. dubitatum is highly specific to capybaras, and is only rarely found on other host species, including humans (GUGLIELMONE et al., 2006;LABRUNA et al., 2007;NAVA et al., 2010).
In Brazil, A. cajennense is the main vector of the bacterium Rickettsia rickettsii, the etiological agent of Brazilian spotted fever (BSF), which is the most lethal rickettsiosis in the world (LABRUNA, 2009).Recent studies have reported that most of the BSF-endemic areas contain large populations of free-living capybaras, which act as primary hosts for both A. cajennense and A. dubitatum, and are exposed to rickettsial infection (HORTA et al., 2007;GUEDES;LEITE, 2008;PEREZ et al., 2008;SOUZA et al., 2008).In addition, it has been demonstrated that capybaras play an important role in the epidemiology of BSF, since they serve as a rickettsial source for creating new lineages of R. rickettsii-infected A. cajennense ticks (SOUZA et al., 2009).
The role of A. dubitatum in the epidemiology of BSF remains unknown.Regardless of being a common tick species in many BSF-endemic areas, A. dubitatum has never been found infected with R. rickettsii.Nevertheless, 6 to 45% of A. dubitatum ticks have been found to be infected by Rickettsia bellii (a rickettsia of unknown pathogenicity for humans) in many tick populations, including some from BSF-endemic areas (LABRUNA et al., 2004;HORTA et al., 2007;PACHECO et al., 2009).In addition, one study reported that 7.5% of the A. dubitatum ticks from a BSFendemic area of São Paulo were infected by the Rickettsia sp.strain Cooperi, which is possibly a new strain of the human pathogen Rickettsia parkeri (LABRUNA et al., 2004;LABRUNA, 2009).
The goal of the present study was to investigate rickettsial infection in A. cajennense and A. dubitatum ticks collected in a BSF-endemic area of the state of Minas Gerais, Southeastern Brazil.In this same area, Guedes et al. (2005) reported that 1.28% of the A. cajennense ticks were infected by R. rickettsii, whereas no rickettsial infection was found in A. dubitatum ticks.

Material and Methods
From May 2006 to April 2008, ticks were collected from naturally infested horses, and from the pasture (free-living ticks), by using CO 2 -baited traps in the Embrapa Gado de Leite (Embrapa Dairy Cattle) experimental farm, located at Coronel Pacheco (21°35' S and 43°15' W), state of Minas Gerais, Brazil, as part of another study that evaluated the seasonal dynamics of Amblyomma spp.ticks.Details about the study area and tick collections have been described by Guedes and Leite (2008).This area is inhabited by large groups of free-living capybaras (Hydrochoerus hydrochaeris), and is considered to be an endemic area for BSF, since human cases of the disease were confirmed recently (GUEDES;LEITE, 2008).In addition, a previous study in the same area reported that an A. cajennense specimen was naturally infected by R. rickettsii (GUEDES et al., 2005).
For the present study, the following numbers of free-living ticks were tested for rickettsial infection: 200 adults of A. cajennense; 200 adults of A. dubitatum; 1,000 larvae; and 1,000 nymphs of Amblyomma spp.These immature stages were not identified to species level at the time of the study.At the same time, ticks were collected from horses and tested for rickettsial infection: these included 200 adults of A. cajennense, and 1,000 larvae and 1,000 nymphs of Amblyomma spp., also not identified to species level.While adult ticks were processed individually, larvae and nymphs were processed in pools of 10 and 5 individuals, respectively.
In the laboratory, ticks were subjected to DNA extraction by means of the guanidine isothiocyanate-phenol technique, as previously described (SANGIONI et al., 2005).For this purpose, each adult tick or a larval or nymphal pool was placed in a 1.5 mL microtube containing 150 µL of TE buffer (Tris HCl 10 mmol.L -1 , EDTA 1 mmol.L -1 , pH 7.4), and was homogenized by using a sterile micropestle.Microtubes containing the homogenized, triturated ticks were then vortexed vigorously.Next, 450 µL of guanidine thiocyanate 5 mol.L -1 ) were added to the tube, which was vortexed again and incubated for 10 minutes at room temperature with brief vortexing every 2 minutes.Following this, 100 µL of chloroform was added to the tube, which was inverted several times and left resting for 2 minutes.The tube was centrifuged at 12,000 x g for 5 minutes to separate the aqueous phase, which was transferred to a clean 1.5 mL microtube.Next, 600 µL of isopropanol was added to the aqueous phase (400 µL), which was homogenized by inverting the tube several times and then incubated at -20 °C for 2 to 18 hours.The tube was then centrifuged at 12,000 x g for 15 minutes; the supernatant was discarded, and the pellet was resuspended with 800 µL of 70% ethanol, and then centrifuged at 12,000 x g for 5 minutes; the supernatant was discarded, and the pellet was dried at room temperature and then resuspended with 30 µL of buffer TE.Finally, the microtubes were incubated at 56 °C for 15 minutes to facilitate DNA homogenization and then stored at -20 °C until tested using the polymerase chain reaction (PCR).For every 10 tubes containing ticks, a blank tube was included in the DNA extraction.
Samples were tested individually by means of PCR targeting a 401-bp fragment of the rickettsial gene gltA, using primers CS2-78 and CS2-323 as previously described (LABRUNA et al., 2004).This PCR protocol has been shown to be 100% effective in detecting R. rickettsii-infected ticks (LABRUNA et al., 2008;2011).In each set of reactions, negative control tubes containing water were included, and also a positive control tube containing DNA of the Rickettsia parkeri strain NOD.Ten microliters of the PCR product underwent electrophoresis in 1.5% agarose gel, stained with ethidium bromide, and examined under UV transillumination.Samples that yielded visible amplicons of the expected size from the gltA-PCR were further tested using a second PCR assay targeting a 532-bp fragment of the rickettsial gene ompA, using the primers Rr-190.70p and Rr-190.602n,as previously described (REGNERY et al., 1991), and using a third PCR assay targeting a 862-bp fragment of the rickettsial gene ompB, using the primers 120-M59 and 120-807, as previously described (ROUX; RAOULT, 2000).All PCR amplicons of the expected size were subjected to direct DNA sequencing in an automated ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA).The BLAST program (National Center for Biotechnology Information, Bethesda, MD, USA) was used to compare appropriate similarities among the rickettsial partial sequences generated in the current study.

Results
None of the ticks collected from horses or the A. dubitatum free-living adult ticks yielded rickettsial DNA by means of PCR (Table 1).However, two free-living A. cajennense adult ticks (1 male, 1 female), both collected in March 2007, and four pools of free-living Amblyomma spp.nymphs (collected in November 2006 andMay, October andNovember 2007) were shown to contain rickettsial DNA using the PCR protocol targeting the rickettsial gene gltA.While the two adult ticks also yielded visible amplicons from both the ompA and the ompB PCR protocol, the four nymphal pools yielded no visible amplicon from the ompA-PCR, and only a weakly visible amplicon of the expected size was yielded from the ompB-PCR.DNA sequences of 321, 490 and 760-bp of the gltA, ompA and ompB products, respectively, from the two A. cajennense adult ticks, were shown to be identical to each other.Moreover, after Blast analysis, they were shown to be all 100% identical to corresponding sequences in the genome of the R. rickettsii strain Sheila Smith (GenBank accession number CP000766).DNA sequences of 350-bp obtained from the gltA-PCR products of the four nymphal pools of Amblyomma spp.were identical to each other, and after Blast analysis, were shown to be closest (99.4%; 348/350) to the corresponding sequence of R. tamurae (AF394896), and second closest (99.1%; 347/350) to Rickettsia monacencis (DQ100163).No DNA sequence of quality was obtained from the ompB-PCR performed on these four Amblyomma spp.nymphal pools.
The gene partial sequences obtained in the present study have been deposited in GenBank under the accession numbers JF690667 (gltA), JF690668 (ompA) and JF690669 (ompB) for R. rickettsii, and JF690670 for the gltA partial sequence generated from the four Amblyomma spp.nymphal pools.

Discussion
The area of the present study has been considered to be endemic for BSF because of the recent history of BSF cases in humans (GUEDES;LEITE, 2008).Therefore, our findings of two R. rickettsii-infected A. cajennense ticks corroborate the endemic status.Guedes et al. (2005) found that 1.28% of the A. cajennense ticks collected from this same area during 2003-2004 were infected by R. rickettsii.In the present study, the R. rickettsii-infected ticks were collected in March 2007; thus, it is likely that R. rickettsii infection had been established in this A. cajennense population for at least three years.Considering that a total of 400 adults of A. cajennense were tested (200 from horses and 200 free-living) for rickettsial infection, we found an overall R. rickettsii infection rate of 0.5% in this tick species.Although this rate is lower than what was reported by Guedes et al. (2005), it is within the values reported for other tick vectors of R. rickettsii, which ranged from 0.05 to 1.3% (BURGDORFER, 1988;PINTER;LABRUNA, 2006).
No rickettsial infection was detected in A. dubitatum adult ticks.Although this result was in accordance with Guedes et al. (2005), who also found no rickettsial infection in A. dubitatum from the same area of this study, it contrasts with other studies, especially in the state of São Paulo, where the majority of the A. dubitatum populations are infected with R. bellii (HORTA et al., 2007;PACHECO et al. 2009).
We found four Amblyomma nymphal pools to be infected by a new rickettsial strain sharing closest identity (99.4%) with R. tamurae, based on a partial fragment of the gltA gene.Since we detected this rickettsial strain in nymphal pools, we suppose that the agent was infecting nymphs of one of the two Amblyomma species found at the study site, namely A. cajennense or A. dubitatum.Interestingly, this new rickettsial strain has never been reported before, even though hundreds of A. cajennense or A. dubitatum ticks have been tested for rickettsial infection in other areas (GUEDES et al. 2005;SANGIONI et al., 2005;HORTA et al., 2007;PACHECO et al., 2009).Currently, R. tamurae is known to occur in Japan, where it infects the tick Amblyomma testudinarium (FOURNIER et al., 2006).Its role as a human or animal pathogen remains unknown.Unfortunately, we could not amplify fragments of other rickettsial genes of the new rickettsial strain found in Amblyomma nymphs, thus precluding any further taxonomic inference from our findings.Further studies are needed to determine the tick species that carry this new rickettsial strain, which should be isolated from ticks in order to provide suitable molecular characterization and thus reach conclusions regarding its taxonomic position.

Table 1 .
Rickettsia infection, as determined using molecular methods applied to Amblyomma spp.ticks that were collected from horses and vegetation (free-living) in Coronel Pacheco, State of Minas Gerais, Brazil.
*Nymphs were tested in 200 pools, each containing 5 ticks.Four pools were found to be infected, which represents a minimal infection rate of 0.4%, assuming that each PCR-positive pool contained only one infected tick.**This Rickettsia sp. was genetically closest to Rickettsia tamurae.