MOLECULAR DETECTION OF BARTONELLA QUINTANA, B. ELIZABETHAE, B. KOEHLERAE, B. DOSHIAE, B. TAYLORII, AND RICKETTSIA FELIS IN RODENT FLEAS COLLECTED IN KABUL, AFGHANISTAN

0 naire Interarme es , BP 05, 31998 Toulouse Arme es , France 1 PIERRE-EDOUARD FOURNIER , JEAN-MARC ROLAIN, SEBASTIEN BRIOLANT, BERNARD DAVOUST The prevalences of Bartonella spp. and Rickettsia spp. were investigated using molecular methods in 77 rodent fleas collected in November 2002 by the French forces detachment in Kabul, Afghanistan. Overall, Bartonella DNA was detected in 15.5% of gerbil fleas and 40.5% of rat fleas, whereas Rickettsia felis was found in 9% of gerbil fleas. We described for the first time in this country Bartonella quintana, B. koehlerae, B. taylorii, and Rickettsia felis in fleas from the gerbil species Meriones lybicus, and B. elizabethae and B. doshiae in rat fleas. Of these, B. quintana, B. elizabethae, B. koehlerae, and R. felis are recognized human pathogens. These results emphasize the potential risk of flea-borne infections transmitted by rodents in this area, and suggest that preventive measures should be taken in the general framework of zoonoses management. - Since the end of 2001, French forces have taken part in a multinational operation in Afghanistan that involves more than 20 different countries. In the French camp in Kabul, Afghanistan, high rate of rodent infestation was observed. Infections caused by Bartonella spp. and Rickettsia spp. occur worldwide and can cause severe human diseases. Most of these infections are transmitted from their animal reservoir to humans via arthropod ectoparasitic vectors such as ticks, fleas, and lice.1 Several studies have highlighted that Bartonella spp. are abundant in wild rodent populations2,3 and in rodent fleas.4 In Asia, pathogenic Bartonella spp. have been reported in rodents,2,5 and Rickettsia felis has previously been detected in mammal fleas.6 In addition, fleas have been shown to be competent vectors of at least some rodent Bartonella7 and Rickettsia8 species. Since few epidemiologic data are available in the international literature regarding zoonoses in Afghanistan, and given the potential risk of zoonosis transmission to military personnel, we investigated the prevalence of Rickettsia spp. and Bartonella spp. in rodent fleas. MATERIALS AND METHODS In November 2002, 55 rodents were captured in the French detachment near the Kabul airport. These rodents included 20 gerbils (Meriones lybicus), 10 rats (Rattus spp.), 23 mice (Mus spp.), and 2 hamsters (Cricetulus migratorius). The animals were caught in the same collection area in close proximity to the tents where the soldiers lived. Captures were made either using home-made traps, allowing rodents to be caught alive, or locally bought traps, which instantly killed them. Rodents were taxonomically identified by using morphologic criteria. We carefully searched for fleas on the rodents shortly after death or anesthesia conducted with ketamine. They were then conserved in sealed, preservative-free, plastic tubes, and were kept frozen at 20C until further processing. Apart from fleas, no other arthropods were found on the rodents. For technical reasons, we only tested the rodent fleas, not the rodents themselves. Identification of the fleas was not carried out at the time of the study. To our knowledge, no description of flea species infesting rodents has been conducted in Afghanistan for more than 30 years. In Asia, Xenopsylla cheopis, the oriental rat flea, is often prevalent,9,10 but as many as 53 different flea species have been described among rodent populations in the same geographic area.11 Fleas were transported to Marseille, France and washed in phosphate-buffered saline for 72 hours at 4C. They were subsequently rinsed in distilled water, dried on sterile filter paper, and crushed. The DNA was extracted from individual fleas by using the QIAamp tissue kit (Qiagen, Hilden, Germany) following the manufacturers instructions. Rickettsial DNA was detected by a polymerase chain reaction (PCR) using primers specific for the citrate synthase gene (gltA), a gene present in most Rickettsia species.12 The primers RpCS.877p and RpCS.1258 amplified a 396-basepair fragment of gltA.12 Bartonella spp. DNA was detected using the QHVE1 and QHVE3 primers, which amplify a fragment of variable size, depending on species, of the 16S-23S ribosomal RNA spacer (ITS).13 The PCR detection of Bartonella spp. and Rickettsia spp. was conducted separately, with each assay using genus-specific primers. The PCRs were carried out in a Peltier model PTC-200 thermal cycler (MJ Research, Inc., Watertown, MA). Each 25- L reaction mixture contained 1 L of a 1 pmol/ L solution of each primer, 0.5 L of Elongase polymerase (GIBCO-BRL, Gaithersburg, MD), 2.5 L of a solution containing 20 mM of each of dATP, dCTP, dGTP, and dTTP (Invitrogen, Carlsbad, CA), 1 L of elongase buffer A, 4 L of elongase buffer B, 7.5 L of sterile distilled water, and 7.5 L of DNA from fleas. Apart from primers, reaction mixtures were identical for both PCR assays. The following amplification program was used: an initial denaturation for three minutes at 94C, followed by 44 cycles of denaturation at 94C for 30 seconds, annealing at a temperature depending on the primers used for 30 seconds,12,13 and extension at 68C for 90 seconds. At the end of the last cycle, extension was completed by incubation at 68C for 7 minutes. Each PCR included negative (distilled water) and positive (DNA extracted from R. montanensis isolate 2-4-6 as described for Rickettsia amplification and from B. elizabethae for Bartonella amplification) controls. The PCR products were visualized after electrophoresis on a 1% agarose gel. Rickettsial PCR products were sequenced using the primer pairs CS1d-CS409d and CS535d-RpCS.1258 as previously described.12 Bartonella spp. PCR products were sequenced using PCR primers. The PCR products were purified using the QIAquick PCR purification kit (Qiagen) in accordance with the manufacturers recommendations. Sequence reactions from PCR-positive products were carried out using the d Rhodamine Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Warrington, United Kingdom) in an ABI 3100 PRISM (Applied Biosystems) automated sequencer. Sequences were identified by comparison with sequences present in GenBank using the BLAST software.14 A total of 77 fleas were collected and tested (Table 1). Sixty-five percent (13 of 20) of the gerbils were infested with fleas, which included 100% (6 of 6) of the females and 50% (7 of 14) of the males. Seventy percent (7 of 10) of the rats were infested with fleas, which included 71% (5 of 7) of the males and 33% (1 of 3) of the females. The number of fleas collected on each individual animal ranged from 0 to 9. The mean number of fleas per infested rodent was 3.5 on gerbils and 4.6 on rats. In contrast, no fleas were found on the 23 mice and 2 hamsters. As determined by ITS gene sequencing, Bartonella spp. DNA was found in 26% (20 of 77) of the fleas, which included 15.5% (7 of 45) in gerbil fleas and 40.5% (13 of 22) in rat (...truncated)