B. rochalimae in Raccoons, Coyotes, and Red Foxes | CDC EID

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Volume 15, Number 12–December 2009
Dispatch
Bartonella rochalimae in Raccoons, Coyotes, and Red Foxes
Jennifer B. Henn, Bruno B. Chomel, Henri-Jean Boulouis, Rickie W. Kasten, William J. Murray, Gila K. Bar-Gal, Roni King, Jean-François Courreau, and Gad Baneth
Author affiliations: Napa County Health and Human Services, Napa, California, USA (J.B. Henn); University of California, Davis, California, USA (B.B. Chomel, R.W. Kasten); École Nationale Vétérinaire d'Alfort, Maisons-Alfort, France (H.-J. Boulouis, J.-F. Courreau); San José State University, San José, California, USA (W.J. Murray); Hebrew University of Jerusalem, Rehovot, Israel (G.K. Bar-Gal, G. Baneth); and Nature Parks Authority, Jerusalem, Israel (R. King)

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Abstract
To determine additional reservoirs for Bartonella rochalimae, we examined samples from several wildlife species. We isolated B. rochalimae from 1 red fox near Paris, France, and from 11 raccoons and 2 coyotes from California, USA. Co-infection with B. vinsonii subsp. berkhoffii was documented in 1 of the coyotes.

Twelve Bartonella species/subspecies have been recognized as zoonotic agents (1,2), including B. rochalimae isolated from a woman who had traveled from the United States to Peru before becoming ill with fever, splenomegaly, mild anemia, and rash (3). B. rochalimae, previously described as a B. clarridgeiae–like organism, has also been isolated from rural domestic dogs and gray foxes (Urocyon cinereoargenteus) from northern California (4,5). A case of fatal endocarditis in a domestic dog was associated with a B. clarridgeiae–like strain (6), later determined to be identical to B. rochalimae (5). Recently, B. rochalimae DNA was detected in a dog from Greece (7). High (43%) prevalence of bacteremia observed in gray foxes in California suggests that they might act as a wildlife reservoir for this newly identified species. Furthermore, several B. clarridgeiae–like and B. rochalimae genes have been detected in fleas collected from humans (8), rodents (9,10), red foxes (Vulpes vulpes) (11), and the environment in the Democratic Republic of Congo (12) during a plague outbreak. To determine whether other wildlife reservoirs exist, we tested samples from 3 additional wildlife species: coyotes, raccoons, and red foxes.

The Study
From 1996 through 1999 in central coastal California, 21 Canis latrans coyotes (3 juveniles [<1 year of age] and 18 adults) and 42 Procyon lotor raccoons (11 juveniles and 31 adults) were trapped. In 2002, a blood sample was collected from a road-killed red fox near Paris, France. All samples were collected in EDTA tubes and frozen at -70°C until plated on heart infusion agar containing 5% rabbit blood and incubated in 5% CO2 at 35°C for up to 4 weeks (13); subsequently, extracted DNA was tested for Bartonella spp. by PCR. In addition, from May 2003 through September 2004, blood was collected from 42 red foxes (23 females and 19 males; 2 kits [<1 year] and 40 adults) in Israel, and extracted DNA was tested for Bartonella spp. by PCR.

Bartonella isolates from 2 (9.5%) coyotes (coyote 004 [7-month-old male] and coyote 22 [adult female captured in central California], which yielded 2 different-size colonies: coyote 22/sub1, large size; coyote 22/sub2, small size), 11 (26%) of the raccoons (7 adult females and 4 adult males), the 1 (100%) red fox from France, and DNA from the blood of 2 (5%) foxes from Israel were compared with B. rochalimae strains isolated from a human, rural dogs, and gray foxes. Bartonella isolates were analyzed by PCR restriction fragment length polymorphism (RFLP) of the 16S–23S intergenic transcribed spacer (ITS) region (all strains) and the gltA, rpoB and ftsZ genes (raccoons, gray foxes, coyotes, and dogs), as previously described (5). For the isolate from the red fox from France, extracted DNA was also amplified for fragments of the groEL gene by using the primer sets HSPps1, HSPps2, and HSPsp4 (11,14). Sequencing was done in both directions by using a fluorescence-based automated sequencing system (Davis Sequencing, Davis, CA, USA). Sequences were imported into Vector NTI Suite 9.0 software (Invitrogen, Carlsbad, CA, USA) to obtain a consensus sequence. Align X in Vector NTI was used for aligning sequence variants with each other and other known Bartonella spp. for each of the 4 genes. A neighbor-joining tree was constructed in MEGA version 3.0 (www.megasoftware.net) by concatenating the 4 sequences. Bootstrap replicates were performed to estimate node reliability of the phylogenetic tree; values were obtained from 1,000 randomly selected samples of the aligned sequence data. Sequence data for the groEL gene of the isolate from the fox in France (GenBank accession no. FJ545656) was compared with sequences of DNA extracted from fleas collected on 4 foxes from Hungary (11) and deposited in GenBank under accession no. DQ522300.

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