Published Ahead of Print 24 December 2008.
2009, 47(3):787. DOI:
J. Clin. Microbiol.
Mark D. Kittleson, William P. Thomas and Bruno B. Chomel
Richard N. Brown, Jane E. Koehler, Kristin A. MacDonald,
Jennifer B. Henn, Mourad W. Gabriel, Rickie W. Kasten,
with Isolates from Dogs, Gray Foxes, and a
Phylogenetic Relationship of the
Infective Endocarditis in a Dog and the
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JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2009, p. 787–790
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 3
Infective Endocarditis in a Dog and the Phylogenetic Relationship of
the Associated “Bartonella rochalimae” Strain with Isolates
from Dogs, Gray Foxes, and a Human?
Jennifer B. Henn,1Mourad W. Gabriel,2Rickie W. Kasten,3Richard N. Brown,4Jane E. Koehler,5
Kristin A. MacDonald,6Mark D. Kittleson,6William P. Thomas,6and Bruno B. Chomel3*
Napa County Health and Human Services, Public Health Division, 2344 Old Sonoma Rd., Bldg. G, Napa, California 945591; Comparative
Pathology Graduate Group, University of California, Davis, Davis, California 956162; Department of Population Health and
Reproduction, School of Veterinary Medicine, University of California, Davis, Davis, California 956163; Department of
Wildlife, Humboldt State University, Arcata, California 955214; Division of Infectious Diseases, University of California,
San Francisco, San Francisco, California 941435; and Department of Medicine and Epidemiology, School of
Veterinary, Medicine, University of California, Davis, Davis, California 956166
Received 15 July 2008/Returned for modification 11 November 2008/Accepted 15 December 2008
The first case of canine endocarditis caused by “Bartonella rochalimae” is reported. By PCR-restriction
fragment length polymorphism, sequence, and phylogenetic analyses, Bartonella isolates from a dog with
endocarditis, 22 gray foxes, and three dogs, described as B. clarridgeiae like, were confirmed to belong to the
new species “B. rochalimae,” suggesting canids as the natural reservoir.
The genus Bartonella comprises more than 20 species and
subspecies, many of which are agents of zoonoses. Bartonella
spp. are usually vector borne, with the vector and reservoir host
varying depending on the Bartonella species involved. How-
ever, the vector has not been definitively identified for many
recently described Bartonella species, nor has the mammalian
reservoir (1). One such new species is “Bartonella rochalimae,”
which was isolated from a human after her visit to Peru (10). It
is believed that this human was an accidental host for “B.
rochalimae,” with the reservoir host not yet identified. Domes-
tic dogs, like humans, appear to be accidental hosts of several
Bartonella species (1, 3, 7) and may serve as sentinels for
human exposure to Bartonella. The first Bartonella species iso-
lated from a dog was B. vinsonii subsp. berkhoffii (5), which
causes most canine Bartonella endocarditis cases (2, 18) and
has been associated with at least one human case of endocar-
ditis (25). Infection of dogs with other Bartonella species, in-
cluding B. clarridgeiae (8), B. washoensis (9), B. henselae (15),
B. elizabethae (19), and B. quintana (14), has also been docu-
mented. Infection of dogs with Bartonella spp. has been asso-
ciated with endocarditis (5, 8, 14, 16, 18), arrhythmias,
myocarditis (16), peliosis hepatis (15), granulomatous lymph-
adenitis, granulomatous rhinitis (21), and granulomatous and
lymphocytic hepatitis (12).
Case report. A 9-year-old male, neutered shepherd mix from
San Francisco was referred to the University of California,
Davis, Veterinary Medical Teaching Hospital for evaluation of
lameness and obtunded mentation in January 2000. Blood
cultures performed at that time were negative. Echocardiog-
raphy revealed a hyperechoic vegetative lesion on the right
coronary cusp of the aortic valve and severe aortic insuffi-
ciency. The lesion was consistent with severe infective aortic
valvular endocarditis. The dog (dog 318006) died in August
2000, and DNA was extracted from the damaged aortic valve
following necropsy. The Bartonella strain infecting this dog was
identified as closely related to B. clarridgeiae (designated B.
clarridgeiae-like) based on the PCR-restriction fragment length
polymorphism (RFLP) pattern and partial sequencing of the
citrate synthase (gltA) gene. The B. clarridgeiae antibody titer
was determined by immunofluorescence to be 1:1,024. Further
identification of the infecting species was not undertaken at the
To further identify this canine strain, Bartonella DNA se-
quences amplified from the damaged valve were compared
with DNA sequences from Bartonella isolates previously found
to be closely related to B. clarridgeiae by sequence analysis (10,
13). Isolates used for comparison included B. clarridgeiae-like
isolates from 22 gray foxes (Urocyon cinereoargenteus) and
three dogs, described in a previous study (13), and an isolate
from a woman with a history of travel to Peru before the onset
of her febrile illness. The Bartonella species infecting the
woman was recently described and designated “B. rochalimae”
sp. nov. (10).
PCR-RFLP analysis of the internal transcribed spacer (ITS)
region (24) and the gltA (20), rpoB (23), and ftsZ (26) genes
was performed on DNA extracted from the damaged canine
heart valve, as well as on the fox, dog, and human isolates.
DNA extraction and PCR vials were set up as previously de-
scribed (6, 13). The primers for the rpoB gene were 5?-CGCA
TTGGCTTACTTCGTATG-3? and 5?-GTAGACTGATTAG
AACGCTG-3?. The PCR conditions were 94°C for 10 min; 35
cycles of 94°C for 0.5 min, 53°C for 0.5 min, and 72°C for 1 min;
and 72°C for 5 min. The primers for the ftsZ gene were 5?-A
TTAATCTGCAYCGGCCAGA-3? and 5?-ACVGADACAC
GAATAACACC-3?. The PCR conditions were 94°C for 10
min; 44 cycles of 94°C for 0.5 min, 55°C for 0.5 min, and 72°C
* Corresponding author. Mailing address: Department of Popula-
tion Health and Reproduction, School of Veterinary Medicine, Uni-
versity of California, Davis, Davis, CA 95616. Phone: (530) 752-8112.
Fax: (530) 752-2377. E-mail: firstname.lastname@example.org.
?Published ahead of print on 24 December 2008.
on September 15, 2012 by UC DAVIS SHIELDS LIBRARY
for 1 min; and 72°C for 10 min. The primers and PCR condi-
tions for the 16S-to-23S ITS region and the gltA gene were
previously described (13). An approximately 400-bp fragment
of the gltA gene, an 860-bp fragment of the rpoB gene, a 900-bp
fragment of the ftsZ gene, and a 670-bp fragment of the ITS
region were amplified and then verified by gel electrophoresis.
The amplified product of the gltA gene was digested with the
restriction endonucleases TaqI (Promega, Madison, WI), HhaI
(New England BioLabs), AciI (New England BioLabs), and
MseI (New England BioLabs). The restriction endonucleases
HhaI (New England BioLabs) and AluI (Promega, Madison,
WI) were used for digestion of the amplified product of the
rpoB gene. Finally, the amplified product of the 16S-to-23S ITS
region was digested with the restriction endonuclease HaeIII
(Promega, Madison, WI). Banding patterns were compared
with B. vinsonii subsp. berkhoffii (ATCC 51672), B. henselae
(strain U-4; University of California, Davis), and B. clarridgeiae
PCR products from the gltA, rpoB, and ftsZ genes and the
ITS region were purified with the QIAquick PCR purification
kit (Qiagen Sciences, Germantown, MD), and both strands
were sequenced with a fluorescence-based automated sequenc-
ing system (Davis Sequencing, Davis, CA). Raw sequence data
were imported into Vector NTI Suite 9.0 software (Invitrogen
Co.). A consensus sequence from both strands was obtained
and compared with nucleic acid sequence entries in GenBank
by using BLASTn (http://ncbi.nih.gov/BLAST/). Sequence
variants for each separate gene and for a concatenated se-
quence of the four gene fragments were then aligned with each
other and with relevant sequences from the GenBank database
by using AlignX in Vector NTI. A percent similarity table
comparing pairs of sequences was also generated after multiple
alignments. Phylogenetic trees were constructed for each gene
by using both the neighbor-joining and maximum parsimony
methods in MEGA version 3.0 (http://www.megasoftware.net)
(17). Bootstrap replicates were performed to estimate the
node reliability of the phylogenetic trees, with values obtained
from 1,000 randomly selected samples of the aligned sequence
PCR amplification of the ITS region and the gltA, rpoB, and
ftsZ genes produced fragments strongly suggestive of Bar-
tonella. The molecular banding patterns of all of the isolates
and the endocarditis dog strain were identical to each other
based on RFLP analysis of the gltA gene, the rpoB gene, and
the ITS region. Because the RFLP profiles were identical for
the 22 gray fox isolates, only 6 isolates from this group were
selected for sequencing of the gltA, rpoB, and ftsZ genes and
the ITS region. Parts of these four sequences were also ob-
tained from the three dogs, the canine endocarditis case, and
the human “B. rochalimae” isolate. A BLAST search with
partial sequences from the ITS region revealed a high degree
of similarity with a GenBank entry (accession number
AF415211.1) for a nucleic acid sequence from a flea (Pulex sp.)
collected from a person in Peru (22), also reported to be the
closest to the human isolate of “B. rochalimae” (10), followed
by the sequence of B. clarridgeiae. Sequence data from the
Peruvian flea are not available for the gltA, rpoB, and ftsZ
genes. The levels of similarity between the Bartonella strains in
this study and B. clarridgeiae ranged from 95.5% for the gltA
gene to 92.3%, 94.3%, and 76.2% for the rpoB gene, the ftsZ
gene, and the ITS region, respectively (Table 1). Small differ-
ences (1 to 4 bp) were observed between two or more genes
when the northern California dog and fox isolates were com-
pared with those from the dog with endocarditis and with the
human “B. rochalimae” isolate. The ITS sequence from the
human “B. rochalimae” isolate was identical to the sequence
from the Peruvian flea that was also the closest match to the
dog and gray fox Bartonella ITS sequences. No differences
were found among or between the rural dog and gray fox
isolates from northern California for the four gene fragments.
Therefore, a representative sequence was chosen from one of
the dog (dog 131) and one of the fox (fox 008) isolates for
inclusion in the phylogenetic analysis (Fig. 1). The tree, con-
structed from a merged set of concatenated sequences, showed
that the dog and gray fox strains cluster together in a distinct
group that includes the human “B. rochalimae” isolate and is
closely related to B. clarridgeiae (Fig. 1). A tree was also con-
structed by using only ITS sequences from the “B. rochalimae”
strains, B. clarridgeiae, and B. vinsonii subsp. berkhoffii in order
to demonstrate that the Pulex sp. sequence from Peru groups
with the human “B. rochalimae” isolate, while the dog and gray
fox samples from California cluster together (Fig. 2).
Discussion. We recently reported the isolation of B. clar-
ridgeiae-like strains from 22 gray foxes and three domestic dogs
from northern California (13). However, PCR-RFLP and se-
quence analyses of the gltA, rpoB, and ftsZ genes and the
16S-to-23S ITS region of these B. clarridgeiae-like isolates and
from the DNA extracted from the damaged cardiac valve of a
dog with endocarditis confirmed that all of these strains belong
to the new species “B. rochalimae,” when compared to the only
human isolate of “B. rochalimae” (?99.5% similarity). “B.
rochalimae” was isolated from an American woman who pre-
sented with fever, rash, and splenomegaly (10). She had a
history of travel to Peru, where she sustained multiple insect
bites. This is the first time that “B. rochalimae” has been
identified in domestic and wild animals and the first report of
“B. rochalimae” isolation from mammals in North America.
The results of this study suggest that this new species of Bar-
tonella is zoonotic and that it could occur in both wildlife
species and domestic dogs in areas within and outside of Cal-
ifornia. It is also the first time that this new Bartonella species
has been associated with a case of endocarditis, as previously
reported in humans and dogs for many other Bartonella species
(7). It is not unexpected that this new species of Bartonella is
associated with disease in both dogs and humans, because dogs
TABLE 1. Levels of similarity between B. clarridgeiae and new
“B. rochalimae” strains in the citrate synthase (gltA), the RNA
polymerase beta subunit (rpoB), the cell division protein
(ftsZ), and the 16S-to-23S intergenic spacer (ITS) region
% Similarity to B. clarridgeiae
Humboldt County dogs,
aIsolated designated “B. rochalimae.”
788NOTESJ. CLIN. MICROBIOL.
on September 15, 2012 by UC DAVIS SHIELDS LIBRARY
have been reported to become infected mainly by species of
Bartonella known to be human pathogens and exhibit many of
the same clinical manifestations (4). Endocarditis appears to
be the most dramatic clinical expression of Bartonella infection
in dogs (1, 2, 5, 7, 16), and the spectrum of Bartonella species
or subspecies involved is widening (8, 9, 18). The canine en-
docarditis case included in this study is strongly suspected to
have been caused by this newly identified Bartonella species.
Factors supporting causation include a failure to isolate other
bacteria by conventional blood culture and PCR amplification
of the Bartonella DNA characterized above from the abnormal
aortic valve, as well as a high Bartonella antibody titer (?1:512
in the present case) commonly observed in cases of endocar-
DNA sequences from the human “B. rochalimae” isolate and
the Peruvian flea PCR product paired closely together in our
phylogenetic analysis, as did sequences from the dog from
rural northern California and the gray fox isolates from Hum-
boldt County, reflecting small differences in gene sequences by
geographical origin. The vector responsible for the transmis-
sion of this new species of Bartonella is unknown. However, the
finding that DNA sequenced from a Pulex sp. flea collected in
Peru (22) is a close match with ITS sequences from isolates in
California supports a role for fleas as a potential vector. “B.
rochalimae” and B. vinsonii subsp. berkhoffii DNAs have been
identified in fleas (Pulex spp.) collected from the foxes sampled
as part of the present study (11), but further studies are needed
to demonstrate vector competence.
Nucleotide sequence accession numbers. Representative se-
quences from strains analyzed in this study were submitted to
FIG. 1. Phylogenetic tree of Bartonella species based on the combined gltA, rpoB, ftsZ, and ITS sequence alignments. The cluster with dog
318006, the human-associated Bartonella sp., dog 131, and Fox 008 represents the “B. rochalimae” group. The tree shown is a neighbor-joining tree
based on the Kimura two-parameter model of nucleotide substitution. Bootstrap values are based on 1,000 replicates. The human-associated
Bartonella sp. has been designated “B. rochalimae.” The analysis provided tree topology only, and the lengths of the vertical and horizontal lines
are not significant.
FIG. 2. Phylogenetic tree based on the 16S-to-23S intergenic
spacer region showing the grouping of “B. rochalimae” strains from
domestic dogs, gray foxes, a human, and a Pulex sp. flea in relation to
B. clarridgeiae. The human-associated Bartonella sp. has been desig-
nated “B. rochalimae.” The tree shown is a neighbor-joining tree based
on the Kimura two-parameter model of nucleotide substitution. Boot-
strap values are based on 1,000 replicates. The analysis provided tree
topology only, and the lengths of the vertical and horizontal lines are
VOL. 47, 2009NOTES 789
on September 15, 2012 by UC DAVIS SHIELDS LIBRARY
(DQ676484 (dog 131) and DQ676488 (dog 318006); rpoB,
DQ676485 (dog 131) and DQ676489 (dog 318006); ftsZ,
DQ676486 (dog 131) and DQ676490 (dog 318006); 16S-to-23S
ITS, DQ676487 (dog 131) and DQ676491 (dog 318006).
underthefollowing accessionnumbers: gltA,
This work was supported by a grant from the Center for Companion
Animal Health at the University of California, Davis, and by the
American Kennel Club Canine Health Foundation. J.E.K. was sup-
ported by NIH R01 AI43703, R01 AI52813, and a Burroughs Well-
come Fund Clinical Scientist Award in Translational Research.
We thank Jerry Theis, the Hoopa Tribe, and the Rural Area Vet-
erinary Service for their contributions to this research.
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790NOTES J. CLIN. MICROBIOL.
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