Rickettsia felis and Bartonella spp. in Fleas from Cats in Albania

Introduction

K nowledge of the epidemiology of vector-borne diseases in Albania is scarce. As in other countries in Europe, free-roaming cat populations exist. Previous studies have shown that the cats in Albania are frequently infested with fleas (Knaus et al. 2009, Xhaxhiu et al. 2009). Fleas can serve as vectors for zoonotic bacterial pathogens like Bartonella (B.) and Rickettsia (R.) species, which have been isolated worldwide (Bitam et al. 2010). For example, Rickettsia felis, an obligate intracellular bacterium, which may cause spotted fever rickettsiosis in humans, was first described as a human pathogen in the early 1990s (Schriefer et al. 1994) and has since been reported in patients worldwide (Bitam et al. 2010). Bartonella henselae, a facultative intracellular bacterium, is known to cause cat-scratch disease (CSD) in humans, a febrile disease associated with lymphadenopathia (Bitam et al. 2010). In some cases of CSD, Bartonella clarridgeiae was suspected to be involved as specific antibodies to that organism had been detected (Chomel et al. 2006). The aim of this article was to report data on the presence of Rickettsia and Bartonella species in fleas from Albania.

Materials and Methods

For this study, fleas collected from 78 free-roaming cats from suburban areas of Tirana, Albania, and identified to species (Jancke 1938) were used. DNA of individual fleas was extracted using the QIAamp^® DNA MiniKit (Qiagen, Hilden, Germany) after homogenization of the fleas in a TISSUELYSER (Qiagen). Quality and quantity of extracted DNA was evaluated with NANODROP^® ND-1000 (PEQLAB, Erlangen, Germany). PCR detection of Rickettsia spp. (gltA gene) was carried out under conditions described previously (Regnery et al. 1991, Silaghi et al. 2008). For the detection of Bartonella spp., a PCR targeting the 16S-23S rRNA intergenic region and discriminating six different pathogenic Bartonella species by fragment length was used (Jensen et al. 2000). The High Fidelity Expand plus Polymerase (Roche, Mannheim, Germany) was used for Rickettsia PCR and the HotMaster™ Taq DNA Polymerase (5PRIME, Hamburg, Germany) for the Bartonella PCR in a Mastercycler^® gradient (Eppendorf, Hamburg, Germany). PCR products were purified with the QIAquick^® PCR purification kit (Qiagen) and sequenced at Eurofins MWG Operon (Ebersberg, Germany). Sequence homology search was performed using BLASTn^SM (www.ncbi.nlm.nih.gov.library.vu.edu.au/BLAST/). Exact 95% confidence intervals (CI) were calculated for the infection rates of the fleas with the Clopper and Pearson method.

Results

In total, 376 fleas were collected from the cats (1 to 50 fleas per cat); 371 were identified as the cat flea Ctenocephalides felis and five as the dog flea Ctenocephalides canis. Ten of the 371 C. felis were positive for Rickettsia spp. (infection rate: 2.7%; CI 95%: 1.4–5.0), and 24 of the 371 C. felis were positive for Bartonella spp. (6.5%; CI 95% 4.3–9.5). No DNA of Rickettsia or Bartonella species was detected in any C. canis. In total, fleas from 15 cats (19.2%) were positive for either one or the other of the pathogens. One cat harboured fleas that were infected with both Rickettsia and Bartonella. However, no Rickettsia spp. and Bartonella spp. co-infections were detected in any flea. BLASTn analysis of the sequenced rickettsial PCR products (331 bp) revealed greatest homology (>99%) in GenBank^® with Rickettsia felis URRWXCal2 (GenBank: CP000053). For Bartonella spp., the nucleotide length of the obtained PCR products (154 bp for B. clarridgeiae and 172 bp for B. henselae), and the subsequent sequencing for further confirmation, revealed greatest similarity of the sequence to B. henselae in 22 (>99% similarity, 146 bp sequence compared to B. henselae isolate CE5 16S-23S with GenBank accession no. FJ832091) and B. clarridgeiae in two cases (>99% similarity, 130 bp sequence compared to B. clarridgeiae strain 73 complete genome with GenBank accession no. FN645454). There were no co-infections involving both Bartonella species. Nine cats (11.5%) carried R. felis–infected fleas; six cats (7.6%) carried B. henselae–infected fleas, and one cat (1.3%) carried B. clarridgeiae–infected fleas.

Discussion

To our knowledge, this article is the first report of the molecular detection of R. felis, B. henselae, and B. clarridgeiae in fleas collected from cats from Albania, thus indicating the potential occurrence of the respective diseases in the country. Tirana is inhabited by a large number of cats that often roam freely in proximity to people, which thereby may serve as a potential source of infection for humans.

R. felis and B. henselae/B. clarridgeiae have different modes of transmission (Bitam et al. 2010). For R. felis, the flea is the reservoir, and humans become infected through a bite of the infected fleas (Bitam et al. 2010). The cat is known to be the reservoir for both B. henselae and B. clarridgeiae; cat-to-cat transmission occurs via infected cat fleas or feces of infected fleas. Cats also are the primary vectors of the transmission of Bartonella spp. to humans, for example, through scratches contaminated with flea feces (Breidtschwerdt et al. 2000, Bitam et al. 2010). Infected cats may show varying symptoms, but they may also be asymptomatic (Chomel et al. 2006).

Free-roaming cats usually receive no flea control measures; therefore humans coming into contact with these animals should be aware of the risk of coming into contact with infected fleas. The results of this study provided evidence for the presence of R. felis and Bartonella spp. in Albania. Thus, flea-borne spotted fever and CSD should be considered as differential diagnoses when febrile symptoms are presented, especially after contact with cats or their fleas. Effective treatment applied to domestic cats to control flea populations may minimize the potential for human exposure.