Rickettsia massiliae in ticks removed from humans in Castilla y León, Spain

Abstract

Rickettsia massiliae, a spotted fever group (SFG) rickettsia, was first isolated from Rhipicephalus sanguineus collected in Marseille in 1992 [1]. Since then, this rickettsia has been commonly detected by molecular tools in Rhipicephalus ticks from France, Greece, Portugal, Switzerland, Spain, Central Africa and Mali [2]. Recently, R. massiliae has been reported as a human pathogen [3]. This strain was first isolated in 1985 from a Sicilian patient who was hospitalized for fever and a rash. He presented with a necrotic eschar on his right ankle, a maculopapular rash on his palms and soles, and slight hepatomegaly. The strain was stored for 20 years and definitively identified as R. massiliae in 2005. From 1997 to 2003, as part of a currently ongoing study, we analyzed 4,049 ticks that had been removed from 3,685 asymptomatic patients seen for tick bites at hospitals in the region of Castilla y Leon (northwestern Spain). All of these ticks were sent to our laboratory for specific identification and for PCR analysis to look for DNA of Anaplasma phagocytophilum, Borrelia burgdorferi, Francisella tularensis and Rickettsia spp. In this way, we found R. massiliae not only in Rhipicephalus ticks but also in Ixodes ricinus ticks. The 4,059 ticks examined belonged to 14 ixodid and one argasid species. After tick identification, DNA from each individual tick was extracted and analysed by PCR. Rickettsial DNA was detected in these tick samples with primers specific for rickettsiae (i.e., RpCS.877-RpCS.1258, which amplify a 380–397 bp fragment of the gltA gene, and Rr190.70-Rr190.701, which amplify a 629–632 bp fragment of the ompA gene), as described elsewhere [4, 5]. The rickettsiae detected were then identified by sequencing the amplified fragments and comparing the sequences obtained with those contained in gene databases. DNA contamination and carry-over of amplified products were prevented by using sterile tools at all times and performing each step of the analysis in separate work areas. Two negative controls (ultrapure water and pathogen-free tick DNA) were included in each amplification trial. These controls never amplified. Among the 4,049 ticks analyzed, a total of 320 (7.9%) ticks belonging to ten species were PCR-positive for rickettsiae, as indicated by the successful amplification of the gltA, the ompA, or both fragments. In 48 of these 320 PCR-positive ticks, namely, 37 Rhipicephalus turanicus, six R. sanguineus, four I. ricinus and one Rhipicephalus pusillus, the sequences of the gltA amplicons obtained were 99.71–100% identical to the gltA of R. massiliae/Bar29 (GenBank, RMU59719/RSU59720). Additionally, in 40 of these 48 ticks that were gtlA-positive for R. massiliae/Bar 29 (30 R. turanicus, six R. sanguineus and four I. ricinus ticks), the ompA amplicon was also obtained. Due to reasons as yet unknown, the ompA amplification failed in Eur J Clin Microbiol Infect Dis (2006) 25:811–813 DOI 10.1007/s10096-006-0217-9