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Emerging Infectious Diseases

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Acknowledgments
References
Table
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Volume 14, Number 12–December 2008

Letter

Bartonella spp. and Rickettsia felis in Fleas, Democratic Republic of Congo

Cherilyn Sackal, Anne Laudisoit, Michael Kosoy, Robert Massung, Marina E. Eremeeva, Sandor E. Karpathy, Kristen Van Wyk, Elizabeth Gabitzsch, and Nordin S. Zeidner
Author affiliations: Centers for Disease Control and Prevention, Fort Collins, Colorado, USA (C. Sackal, M. Kosoy, K. Van Wyk, N. Zeidner); University of Antwerp, Antwerp, Belgium ( A. Laudisoit); University of Liege, Liege, Belgium (A. Laudisoit); Centers for Disease Control and Prevention, Atlanta, Georgia, USA (R. Massung, M.E. Eremeeva, S.E. Karpathy); and Etubics Corporation, Seattle, Washington, USA (E. Gabitzsch)

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To the Editor: Bartonella and Rickettsia species are pathogens of humans and domestic mammals that may be transmitted by fleas and other arthropods. Rickettsia felis causes flea-borne spotted fever in humans who come into contact with flea-infested domestic and peridomestic animals; worldwide distribution of this pathogen in ectoparasites and mammals makes it an emerging threat to human health (1,2). Likewise, species of the genus Bartonella are associated with an increasing array of human diseases, including trench fever, cat-scratch disease, and endocarditis in immunocompetent patients, and bacillary angiomatosis and peliosis hepatitis in immunocompromised patients (3–5). Although Bartonella spp. and R. felis appear to be globally distributed, their presence in the Democratic Republic of Congo (DRC) has not been previously documented.

Off-host Pulex irritans, Tunga penetrans, Ctenocephalides felis strongylus, Echidnophaga gallinacea, and Xenopsylla brasiliensis were collected in the Ituri district of northeastern DRC from March through April 2007, during an investigation of a plague outbreak. Our investigation area was limited to 4 villages: Djalusene and Kpandruma, which had confirmed plague patients, and Wanyale and Zaa, which had several suspect cases.

We collected fleas by using a kerosene lamp hung above a 45-cm diameter tray containing water (7). Captured fleas were identified using a dissecting microscope and standard morphologic keys, sorted into vials by species and locality, and preserved in 70% ethanol (7). Fleas were separated into 193 pools (2–5 fleas per pool), triturated for 10 minutes; the resultant flea triturate was centrifuged at 3,000 rpm for 10 minutes to collect flea tissue. DNA was then obtained by using the DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA, USA).

Bartonella DNA was detected by PCR amplifying a 379-bp fragment of the citrate synthase gene (gltA) (8). For Rickettsia typhi and R. felis, a real-time multiplex PCR assay targeting a conserved fragment of gltA was used (unpub. data). All assays were run in duplicate, and positive and negative controls were included in all assays. Amplicons were purified with the QIAquick PCR purification kit (QIAGEN) and sequenced in both directions by using a BigDye sequencing kit (Applied Biosystems, Foster City, CA, USA) with the same primers used for PCR amplification. Resultant sequences of Bartonella spp. were aligned with MegAlign by using the Clustal algorithm (DNASTAR, Inc., Madison, WI, USA), and compared with reference sequences obtained from GenBank.

Although Yersinia DNA and R. typhi were not detected, 89 of the 193 pools were PCR positive for either Bartonella spp. or R. felis (Table). Using the Microsoft Excel Add-In PooledInfRate software (Redmond, WA, USA; www.cdc.gov/ncidod/dvbid/westnile/software.htm), we calculated an estimated infection rate of 10.72% (95% confidence interval [CI] 8.52–13.31) for R. felis, 3.66% for Bartonella species, and 0.91% (95% CI 0.40–1.78) for both Bartonella spp. and R. felis (Table).

Phylogenetic analysis indicated several Bartonella spp. in fleas that were closely aligned with pathogenic Bartonella spp., including B. vinsonii, Candidatus B. rochalimaea, and B. clarridgeiae (data not shown). Moreover, Bartonella from 3 pools of P. irritans demonstrated only 1.8% to 2.4% divergence to B. vinsonii subspecies arupensis isolated from a human patient in Wyoming, USA. Likewise, sequences of Bartonella from 1 pool of T. penetrans and 1 pool of P. irritans were 100% identical to Bartonella isolated from a Neotoma mexicana wood rat (GenBank accession no. AF110312); a sequence obtained from 1 flea pool of T. penetrans was 100% identical to the gltA Bartonella sequence found in Orchopeas sexdentatus, collected from Neotoma micropus in New Mexico, USA (data not shown). This finding indicates a new Bartonella species with multiple rodent origins and a more ubiquitous global dissemination than previously determined. Our results also demonstrate the previously unreported detection of R. felis in P. irritans, E. gallinacea, X. brasiliensis, and T. penetrans flea species.

This report suggests that Bartonella spp. and R. felis exists in fleas within the DRC. In addition, we report Bartonella spp. and R. felis DNA in T. penetrans fleas and R. felis DNA in E. gallinacea fleas, vectors not previously associated with these pathogens. Co-infections were also observed in T. penetrans, P. irritans, and C. felis fleas, suggesting a common vector or mammalian host shared by R. felis and Bartonella spp. Flea feedings occur intermittently and on potentially different hosts, thus the vectors described here may acquire multiple bacterial strains for transmission to humans (8). Moreover, PCR assays targeting the cytochrome B gene indicated human blood in the flea pools, demonstrating dual infection (data not shown); this finding shows that the flea species recovered are capable of feeding on humans, have a broad host range, and are capable of transmitting disease to humans (9).

Bartonella spp. and R. felis have been detected previously in fleas within northern and sub-Saharan Africa (10). The presence of Bartonella spp. and R. felis in the fleas is important because they were collected in close contact with humans at risk for multiple exposures within households. Our results suggest that both R. felis and Bartonella spp. are prevalent in this region of the DRC and should be included in the differential diagnosis of potential flea-borne infections in this region of sub-Saharan Africa.

Acknowledgments

We thank Brad Biggerstaff and Becky Eisen for statistical analyses used in this study. S.E.K. was supported by The Oak Ridge Institute for Science and Education.

References

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Table

Table. Detection of Bartonella spp. and Rickettsia spp. DNA in fleas collected in the Democratic Republic of Congo