Rickettsia felis and Bartonella clarridgeiae in Fleas from New Caledonia

R ickettsia felis , an emerging pathogen (Perez-Osorio et al. 2008) with a cosmopolitan distribution, has been associated with various species of fleas. The cat flea, Ctenocephalides felis, is currently the only known biological vector of R. felis; however, molecular evidence of R. felis in other species of fleas has been reported (Reif and Macaluso 2009). Few clinical cases have been documented, and the current knowledge on the distribution of the disease is based on the detection of R. felis in fleas. The cat flea is a ubiquitous parasite of many types of domesticated animals, including cats and dogs, and infests the animal companions of humans worldwide. Because it also feeds readily on humans, it is considered as a vector of several important zoonoses, including bartonellosis and spotted fever caused by R. felis (Rolain et al. 2003). The epidemiology of R. felis infection remains poorly known, although R. felis has been identified in fleas in many regions, including Europe (Capelli et al. 2009), Asia (Jiang et al. 2006), North and Sub-Saharan Africa (Bitam et al. 2006, Sackal et al. 2008), and North and South America (Labruna et al. 2007, Reif and Macaluso 2009). Oceania and Pacific region still stay less studied. Recently, it was identified in 15% of the C. felis from New Zealand (Kelly et al. 2004) and in 36% of these fleas in Western Australia (Schloderer et al. 2006) (Fig. 1).

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FIG. 1.

Map of Southern Pacific region with noted places where Rickettsia felis was identified (this study and literature data).

Cats are considered as main reservoir hosts of Bartonella clarridgeiae with worldwide distribution (Chomel et al. 2006). It has been associated with cat-scratch disease in humans and aortic valve endocarditis and hepatic disease in dogs (Chomel et al. 2009). The organism has already been found once in fleas in New Zealand (Kelly et al. 2004) and in domestic cats in Indonesia and the Philippines (Chomel et al. 2009). The prevalence of B. clarridgeiae in cat fleas may be as great as 17% in Europe (Rolain et al. 2003). To the best of our knowledge, no studies of flea-transmitted bacteria pathogenic for humans have been reported previously from Melanesia.

A total of 21 fleas were collected from eight dogs in Païta near Nouméa (New Caledonia, a French Island in Melanesia, see Fig. 1). These were stored in 70% ethanol, transported to France, and identified using a standard taxonomic key (Hopkins and Rothschild, 1966). DNA was extracted from each specimen with QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions and stored at 4°C until use in polymerase chain reaction (PCR) amplifications. Rickettsial DNA was detected by using Rickettsia genus–specific real-time PCR using LightCycler 2.0 equipment and software (Roche Diagnostics GmbH, Mannheim, Germany). Master mixtures were prepared by following the instructions of the manufacturer and with primers and probe targeting a chromosomal gene specific of R. felis bioB, whose sequences specific for R. felis have been described previously (Varagnol et al. 2009). All samples were also screened using a Bartonella genus–specific real-time PCR with a 21-bp probe targeting the 16S/23S RNA gene intergenic spacer (ITS) gene (Varagnol et al. 2009). All PCRs positive in real-time PCR extracts were tested also using a standard PCR with primers designed to amplify a variable-size ITS of Bartonella spp. (Parola et al. 2003) for the species identification. Negative controls used were DNA extracted from laboratory-reared fleas. Positive controls included samples of R. felis and Bartonella elizabethae DNA. DNA sequencing reactions were done for all samples re-amplified in standard PCR for ITS of Bartonella. Data were collected with an ABI Prism 3130xl Genetic Analyzer capillary sequencer (Applied Biosystems, Foster City, CA). Sequences were edited and assembled using Chromas Pro 1.34 (Technelysium Pty. Ltd., Tewantin, Australia). BLAST search was performed for identification of obtained sequence.

All fleas were identified as C. felis. R. felis DNA was detected in 17 (81%) specimens of C. felis. Bartonella DNA was detected using real-time PCR in 1 (5%) flea. Sequencing revealed that the 710-bp product had 100% homology with Bartonella clarridgeiae isolate Houston-2 (AF312497.1).

Our study identified B. clarridgeiae and R. felis in Melanesian cat fleas. The prevalence of R. felis rickettsiosis in humans is unknown but may be high because of the ubiquity of its vector. Human cases have been reported from North America, Europe, Asia, and North Africa (Perez-Osorio et al. 2008), reflecting global emergence of this pathogen. Our data show a high rate (81%) of infected fleas and extend the distribution of the agent of this emerging rickettsiosis. Both frequent possible contact with humans and pathogenicity indicate the emergence of B. clarridgeiae infection as well. The increasing pool of data on distribution and prevalence of these microorganisms in vectors and reservoirs will provide a background for a complex evaluation of their importance for human healthcare.

The islands of Oceania are increasingly visited by tourists; therefore, these data should alert medical workers in Melanesia and around the world regarding the existence of R. felis and B. clarridgeiae in this region.