Presence of Coxiella burnetii in Fleas in Cyprus Anna Psaroulaki, 1 Dimosthenis Chochlakis, 2 Ioannis Ioannou, 3 Emmanouil Angelakis, 1,4 and Yannis Tselentis 2 Abstract Over 40 tick species are naturally infected by Coxiella burnetii. However, little is known about the presence of C. burnetii in other ectoparasites such as fleas. During a 6-year (2000–2006) study, 1147 fleas were collected from 652 animals (252 rats, 118 foxes, and 282 hares) captured from different areas of Cyprus. Three flea species—Xenopsylla cheopis, Ctenocephalides felis, and C. canis—were identified. Fleas were pooled (153 pools) and tested by PCR for the presence of C. burnetii. The pathogen was identified in 25 (16.3%) pools. None of the fleas parasitizing hares was positive for C. burnetii, as opposed to fleas collected from rats (12% pool positivity) and foxes (47.6% pool positivity). The highest prevalence of positive pools was recorded in C. canis (38%) compared to C. felis (16.6%) and X. cheopis (10.8%). All pools of C. canis positive for C. burnetii were removed from foxes (44.4%), whereas all positive X. cheopis (10.8%) were removed from rats. The role of fleas in the maintenance and transmission of C. burnetii among wild vertebrates remains to be determined. Key Words: Coxiella burnetii—Fleas—Cyprus. Introduction Q fever is a zoonosis caused by Coxiella burnetii,a small, obligate, intracellular, Gram-negative bacterium that is prevalent throughout the world (Angelakis and Raoult 2010). It can infect and persist in a wide range of domestic and free-living vertebrates, namely ungulates (although the pathogen is not exclusively associated to them), and ticks. A number of studies have been published on the presence and dispersal of C. burnetii infection in wildlife, whereas over 40 tick species have been found to be naturally infected by C. burnetii. Nevertheless, little is known about the presence of C. burnetii in other ectoparasites, such as fleas. In fact, a single study has been published in the past demonstrating the presence of the pathogen in fleas (Loftis et al. 2006). In Cyprus, a high prevalence of C. burnetii in humans, goats, sheep, and bovines was recorded in past studies (Psaroulaki et al. 2006). Recently, 12.8% of rat collected in different areas of the island revealed antibodies against the pathogen (Psaroulaki et al. 2010). The pathogen has been detected both in endemic and migratory birds, as well as in mouflons and their ticks (Ioannou et al. 2009, 2011). Recent still unpub- lished data of a cross-sectional study have verified the pres- ence of C. burnetii in ticks collected from wildlife. The aim of the present study was to record the presence of C. burnetii in fleas collected from wild animals that may take part in the pathogen’s cycle in Cyprus. Materials and Methods During a 6-year (2000–2006) study, fleas were collected from rats (Rattus norvegicus and R. rattus), foxes (Vulpes vulpes indutus), and hares (Lepus europaeus) captured from 48 different areas of Cyprus. Captures were carried out throughout the year, but mainly in the spring and autumn months. Collected ectoparasites were identified using stan- dard taxonomic keys (Ioannou et al. 2011). Fleas were pre- served in sterile conditions at room temperature before being sent to the Laboratory of Clinical Bacteriology (Q fever reference center) in Heraklion, Crete, Greece. Prior to DNA isolation, each flea was rinsed twice in 70% ethanol and sterile water for 15 min. Fleas were placed in separate ep- pendorf tubes into 200 lL of distilled water, smashed using surgical scissors, and then pooled, with each pool containing three to nine fleas, according to species, host, and region of collection, to a final volume of 200 lL. In some cases, fleas 1 Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Medical School, University of Crete, Heraklion, Greece. 2 Regional Laboratory of Public Health, Heraklion-Crete, Greece. 3 Veterinary services, Nicosia, Cyprus. 4 Aix Marseille Universite ´, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095, Marseille, France. VECTOR-BORNE AND ZOONOTIC DISEASES Volume 14, Number 9, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2013.1399 685