Rickettsia aeschlimannii in Spain: molecular evidence in Hyalomma marginatum and five other tick species that feed on humans. (Letters).

To the Editor: Rickettsia aeschlimannii is a pathogenic spotted fever group rickettsia first isolated from Hyalomma marginatum ticks collected in Morocco in 1997 (1). Later found in H. marginatum ticks from Zimbabwe, Niger, Mali, and Portugal (2), R. aeschlimannii has also been found in a Rhipicephalus appendiculatus tick attached to the right thigh of a patient in South Africa (3). These data suggest a broad geographic distribution for R. aeschlimannii and the possibility that tick species other than H. marginatum may also be suitable vectors for this rickettsia.

The pathogenicity of Rickettsia aeschlimannii in humans has been demonstrated by Raoult et al. (4) in a French patient who became ill after returning from a trip to Morocco. The patient exhibited symptoms similar to those of Mediterranean spotted fever (MSF) produced by R. conorii, with a tache noire--like eschar on his ankle, fever (39.5[degrees]C), and a generalized maculopapular skin rash. The second documented and most recent case of human infection by R. aeschlimannii occurred in a South African man who was bitten by R. appendiculatus; an eschar also developed around the tick attachment site on this patient (3). He was aware of the risk for tick-transmitted disease, so he removed the tick and administered doxycycline; he did not develop additional symptoms.

Over the past 6 years, throughout the region of Castilla y Leon, northwestern Spain, we have collected and identified 3,059 ticks belonging to 15 species (unpub. data) that were attached to persons living in this territory. We have systematically analyzed the ticks by polymerase chain reaction (PCR) to detect those infected with Borrelia burgdorferi, Anaplasma phagocytophila, and Rickettsia spp. This procedure enabled us to identify, for the first time in Spain, R. aeschlimannii in 35 tick specimens belonging to H. marginatum and to another five species.

During the 6-year study, ticks found on patients who sought medical advice in the hospitals and healthcare centers of Castilla y Leon were removed and referred to our laboratory for identification and analysis. Each tick was first disinfected by immersion in 70% alcohol, rinsed in sterile water, and dried on sterile filter paper. We then extracted DNA in 5% Chelex-100, according to the method of Guttman et al. (5). In searching for Rickettsia spp., we proceeded as described by La Scola and Raoult (6): All DNA samples were first tested for a fragment of the rickettsial gltA gene (7), and then, in the gltA-positive samples, a fragment of the rickettsial ompA gene (8) was amplified, sequenced, and compared with gene databases for identification. The gltA amplicon was sequenced only when the ompA was not successfully amplified. To prevent DNA contamination and the carryover of amplified products, we used sterile tools at all times and carried out each step of the analysis (extracting DNA, preparing the reaction mixture, and amplifying and analyzing the PCR product) in separated work areas. Two negative controls (Milli-Q water and DNA from laboratory-reared noninfected ticks) were included in each amplification trial. These controls were never amplified.

We obtained 21 ompA amplicons (629-632 bp) from 21 ticks. One amplicon, from a Haemaphysalis punctata tick, had 100% sequence identity with the ompA of R. aeschlimannii (GenBank accession no. U43800). The nucleotide sequences of the remaining 20 ompA amplicons shared >99% similarity with the ompA of R. aeschlimannii. These 20 amplicons were obtained from nine Hyalomma marginaturn, five Rhipicephalus bursa, three R. turanicus, one R. sanguineus, and two Ixodes ricinus ticks.

In an additional 14 ticks (10 H. marginatum, 2 R. bursa, 1 R. sanguineus, and 1 I. ricinus) we sequenced 14 gltA amplicons (382 bp), which were 100% identical to the gltA of R. aeschlimannii (GenBank accession no. U59722). No other tick-borne pathogens were detected in the 35 R. aeschlimannii-containing ticks.

R. aeschlimannii has never been detected in Spain; therefore, our study constitutes its first citation in this country. Because R. aeschlimannii had been already detected in ticks in Portugal, we believe that its presence in Spain was expected and our finding is not surprising. However, the high number of tick species in which we found this rickettsia was unexpected: six species belonging to four genera. For these six species, the ratio between the specimens infected and the specimens analyzed (as well as the infection rates) were as follows: I. ricinus (3/1,320; 0.23%), H. marginatum (19/324; 5.86%), H. punctata (1/106; 0.94%), R. bursa (7/425, 1.64%), R. sanguineus (2/102; 1.96%), and R. turanicus (3/330; 0.91%). Although H. marginatum was the fourth most anthropophilic species in our study, this species simultaneously showed the highest number of infected specimens and the highest infection rate, making H. marginatum the main vector of R. aeschlimannii in our region. The next most important vectors are Rhipicephalus spp., and in particular R. bursa.

The 35 R. aeschlimannii--positive ticks were removed in the first 6 to 12 hours after attachment, before they could have ingested any blood, thus indicating that they were previously infected with the bacterium. Persons bitten by these specimens had never had symptoms of spotted fever, and they remained asymptomatic after the bite, suggesting that, as expected because of the rapid removal of the ticks, they did not acquire the infection.

Although MSF is endemic in Castilla y Leon (9), we only found one tick infected with R. conorii (0.03%) among the 3,059 analyzed (unpub. data), whereas R. aeschlimannii was much more prevalent in these same ticks (1.14%). Hence, in accordance with what was proposed by Raoult et al. (4) for MSF cases in Morocco, we suspect that many cases of MSF in Castilla y Le6n may really have been due to R. aeschlimannii.

Our findings show that R. aeschlimannii is present in Castilla y Leon, the largest region in Spain, in six tick species that frequently feed on humans. Our observations expand the geographic distribution of this bacterium and the range of its potential tick vectors.

Acknowledgments

We thank Rufino Alamo-Sanz for his invaluable contributions and N. Skinner for revising the English version of the manuscript.

This work was supported by the Consejeria de Sanidad y Bienestar Social of the Junta de Castilla y Leon (Spain).

References

(1.) Beati L, Meskini M, Thiers B, Raoult D. Rickettsia aeschlimannii sp. nov., a new spotted fever group rickettsia associated with Hyalomma marginatum ticks, Int J Syst Bacteriol 1997;47:548-54.

(2.) Parola P, Raoult D. Ticks and tickborne bacterial diseases in humans: an emerging infectious threat. Clin Infect Dis 2001;32:897-928.

(3.) Pretorius A, Birtles RJ. Rickettsia aeschlimannii: a new pathogenic spotted fever group rickettsia, South Africa. Emerg Infect Dis 2002;8:874.

(4.) Raoult D, Fournier PE, Abboud P, Caron F. First documented human Rickettsia aeschlimannii infection. Emerg Infect Dis 2002;8:748-9.

(5.) Guttman D S, Wang PW, Wang IN, Bosler EM, Luft BJ, Dykhuizen DE. Multiple infections of Ixodes scapularis ticks by Borrelia burgdorferi as revealed by single-strand conformation polymorphism analysis. J Clin Microbiol 1996;34:652-6.

(6.) La Scola B, Raoult D. Laboratory diagnosis of rickettsioses: current approaches to diagnosis of old and new rickettsial diseases. J Clin Microbiol 1997;35:2715-27.

(7.) Regnery RL, Spruil CL, Plikaytis BD. Genotypic identification of Rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol 1991; 173:1576-89.

(8.) Roux V, Furnier PE, Raoult D. Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR-amplified DNA of the gene encoding the protein rOmpA. J Clin Microbiol 1996;34:2058-65.

(9.) Boletines Epidemiologicos de Castilla y Leon 1997-2002. (Spain): 2000;16:9-12.

Address for correspondence: Ricardo Perez-Sanchez, Departamento de Patologia Animal, Instituto de Recursos Naturales y Agrobiologia, Consejo Superior de Investigaciones Cientificas, C/ Cordel de Merinas, 40-52, 37007 Salamanca, Spain; fax: [phone]; email: [e-mail]

Pedro Fernandez-Soto, * Antonio Encinas-Grandes, * and Ricardo Perez-Sanche ([dagger])

* Universidad de Salamanca, Salamanca, Spain; and ([dagger]) Instituto de Recursos Naturales y Agrobiologia, Consejo Superior de Investigaciones Cientificas, Salamanca, Spain