Spotted Fever Group Rickettsiae in Ticks Feeding on Humans in Northwestern Spain Is Rickettsia conorii Vanishing? a RICARDO PEREZ-S b ´ ´ ´ PEDRO FERNANDEZ-SOTO, ANCHEZ, c AND ANTONIO ENCINAS-GRANDESa ´ RUFINO ALAMO-SANZ, a Laboratorio
de Parasitolog´ıa, Facultad de Farmacia, Universidad de Salamanca, 37007 Salamanca, Spain b Departamento
de Patolog´ıa Animal, IRNA (CSIC), 37007 Salamanca, Spain
c Direcci´ on General de Salud P´ublica, Consejer´ıa de Sanidad, Junta de Castilla y
Le´on, 47011 Valladolid, Spain
ABSTRACT: During a 7-year study, we identified and analyzed by PCR 4,049 ticks removed from 3,685 asymptomatic patients in Castilla y Le´on (northwestern Spain). A total of 320 ticks (belonging to 10 species) were PCR-positive for rickettsiae. Comparison of amplicon sequences in databases enabled us to identify eigth different spotted fever group (SFG) rickettsiae: Rickettsia slovaca, Rickettsia sp. IRS3/IRS4, R. massiliae/Bar29, R. aeschlimannii, Rickettsia sp. RpA4/DnS14, R. helvetica, Rickettsia sp. DmS1, and R. conorii. Although Mediterranean spotted fever (MSF) is an endemic disease in Castilla y Le´on, R. conorii was found in only one Rhipicephalus sanguineus tick, whereas other pathogenic SFG rickettsiae were much more prevalent in the same area. Our data suggest that in Castilla y Le´on, many MSF or MSF-like cases attributed to R. conorii could have been actually caused by other SFG rickettsiae present in ticks biting people in this region of Spain. KEYWORDS: Mediterranean spotted fever; Rickettsia conorii; R. slovaca; R. aeschlimannii; Castilla y Le´on; Spain
Castilla y Le´on is the largest regional community in the northwestern part of Spain and the largest regional area of Europe. Traditionally, the Mediterranean spotted fever (MSF) caused by R. conorii was thought to be the only prevailing rickettsial disease in this community and the main sources of knowledge are Address for correspondence: Dr. Pedro Fern´andez-Soto, Laboratorio de Parasitolog´ıa, Facultad de Farmacia, Universidad de Salamanca, Avenida Campo Charro s/n. 37007, Salamanca, Spain. Voice: +34-923-294535; fax: +34-923-294515. e-mail:
[email protected] C 2006 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1078: 331–333 (2006). doi: 10.1196/annals.1374.063
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numerous seroepidemiological studies carried out on humans and animals. To date, other rickettsioses have been poorly investigated in this region and it should be useful to expand those investigations, including the molecular identification (and ideally, isolation) of the rickettsiae. The purpose of this study is to report the molecular identification of spotted fever group (SFG) rickettsiae in ticks removed from human subjects in a 7-year study carried out in Castilla y Le´on, and, additionally, to highlight the near absence of R. conorii in ticks from this part of Spain in spite of the frequent MSF cases reported in that area for years. From 1997 to 2003, 4,049 ticks removed from 3,685 asymptomatic patients assisted for tick bites in the hospitals and health care centers of Castilla y Le´on were sent to our laboratory for identification and analysis. Rickettsial DNA was detected by PCR using primers RpCS.877p-RpCS.1258n to target a 380–397-bp gltA fragment and Rr190.70p-Rr190.701n to target a 629–632-bp ompA fragment, as previously described.1 The amplicons obtained were purified, sequenced, and compared in GenBank for identification. The 4,049 ticks identified belonged to 14 ixodid and 1 argasid species. Of the 3,685 patients, 91% were parasitized by a single tick and 9% by two or more. A total of 320 ticks (belonging to 10 species) were PCR-positive for rickettsiae, since in them the gltA, the ompA, or both amplicons were amplified, representing a global infection rate of 7.90%. For unknown reasons, in 72 of the 320 rickettsiae-positive ticks (22.5%) the sequencing of the amplicons failed and, consequently, it was not possible to identify the Rickettsia species involved. In the remaining 248 positive ticks (77.5%), comparison of amplicon sequences in the database enabled us to identify, by order of decreasing abundance, R. slovaca in 59 ticks (58 D. marginatus and 1 D. reticulatus), Rickettsia sp. IRS3/IRS4 in 51 ticks (50 I. ricinus and 1 D. marginatus), R. massiliae/Bar29 in 48 ticks (37 R. turanicus, 6 R. sanguineus, 4 I. Ricinus, and 1 R. pusillus), R. aeschlimannii in 42 ticks (26 H. marginatum, 7 R. bursa, 3 I. ricinus, 3 R. turanicus, 2 R. sanguineus, and 1 H. punctata), Rickettsia sp. RpA4/DnS14 in 35 ticks (25 D. marginatus, 9 D. reticulatus, and 1 I. ricinus), R. helvetica in 8 ticks (8 I. ricinus), Rickettsia sp. DmS1 in 4 ticks (4 D. marginatus) and, finally, R. conorii in 1 tick (1 R. sanguineus). During 1997 to 2003, 353 MSF cases reported in Castilla y Le´on were attributed to R. conorii according to the clinical signs, serological results, and the previous epidemiological data.2 However, in the current study, R. conorii was found in only one R. sanguineus tick. The scarcity of R. conorii is not only unexpected, but also very important from an epidemiological point of view. Recently, we reported the presence of R. aeschlimannii in six tick species that frequently feed on humans in this community, raising the suspicion that many cases of MSF in this region of Spain could have been due to R. aeschlimannii.1 The results presented here show that several pathogenic rickettsiae are much more prevalent than R. conorii in ticks biting people in Castilla y Le´on. This observation suggests that, for years in our community, many MSF or
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MSF-like cases attributed to R. conorii—on the basis of clinical, serological, and epidemiological data—could have been actually caused by other rickettsiae, such as R. aeschlimannii and R. slovaca and, perhaps, by other SFG rickettsiae among those recently identified, whose pathogenicity is not yet undoubtedly proven. The demonstration of this hypothesis requires further genotypic and phenotypic characterization—and ensuing comparison—of both the human and the tick rickettsial isolates.
ACKNOWLEDGMENTS This work was supported by Consejer´ıa de Sanidad, Junta de Castilla y Le´on, and Ministerio de Sanidad y Consumo, Fondo de Investigaci´on Sanitaria (FIS), Red Tem´atica de Investigaci´on Cooperativa EBATRAG-G03/057.
REFERENCES ´ ´ 1. FERNANDEZ -SOTO P., A. ENCINAS-GRANDES & R. P´EREZ-SANCHEZ . 2003. Rickettsia aeschlimannii in Spain: molecular evidence in Hyalomma marginatum and five other tick species that feed on humans. Emerg. Infect. Dis. 9: 889–890. ´ ´ . 1997–2003. Direcci´on General DE CASTILLA Y LEON 2. BOLETINES EPIDEMIOLOGICOS de Salud P´ublica y Consumo. Consejer´ıa de Sanidad. Valladolid, Spain.
