Molecular Detection of Bartonella alsatica in Rabbit Fleas, France

To the Editor: Bartonella alsat-ica was fi rst isolated from the blood of wild rabbits from the Alsace region in France (1). This bacterium is now considered an emerging infectious disease zoonotic agent in persons in close contact with rabbits; at least 2 human cases of endocarditis and 1 human case of lymphadenitis have been reported (2–4). In this study, we report the molecular detection of B. alsatica in fl eas (Spilopsyllus cuniculi) collected from rabbits in southern France. During January and February 2008, a total of 60 fl eas were collected from wild rabbits (Oryctolagus cuniculus) from 3 regions in southern France: Canohes (42°38′N, 2°51′E), Pollestres (42°38′N, 2°52′E), and Toreilles (42°45′N, 2°58′E). The fl eas were collected and identifi ed pheno-typically, kept in ethanol, and sent to Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes in September 2009. DNA from these fl eas, as well as negative controls from uninfected lice maintained as colonies in our laboratory , were extracted by using a QIAmp Tissue Kit (QIAGEN, Hilden, Germa-ny), as described (5). Identifi cation of fl ea species at the molecular level was achieved by PCR amplifi cation and se-quencing of partial siphonapteran 18S rDNA gene (1.95 kbp) as described (5). Sequences were assembled in Sequencher 4.2 (GeneCodes Corporation , Ann Arbor, MI, USA). DNA was used as templates in a real-time quantitative PCR specifi c for a portion of the Bartonella genus 16S–23S intergenic spacer (ITS) performed in a Smart cycler instrument (2). Positive samples at the genus level were confi rmed by PCR amplifi ca-tion and sequencing of the Bartonella ITS region, as described (2). Finally, B. alsatica amplifi cation and specifi c identifi cation was confi rmed by using 2 new specifi c PCRs with primers and TaqMan probes (Applied Biosystems, Courtaboeuf, France) specifi c for a portion of the heat shock protein 60 (hsp60) and the DNA gyrase subunit B (gyrB) genes of B. alsatica (Table). Specifi city of these 2 PCRs was veri-fi ed in silico (computer simulation) and by using a panel of 14 Bartonella species available in our laboratory (data not shown). All fl eas were morphologically identifi ed as S. cuniculi by using current taxonomic criteria (6). Moreover, the 18S rRNA gene amplifi ed and se-quenced as described (6) from fl eas gave a sequence with 100% similarity with the sequence of S. cuniculi fl eas deposited in GenBank …