LETTERS
the exact tissue type of each sample,
only that tonsils, adenoids, or both
combined could be present, the 5.5%
rate we found was about one third the
rate found in tonsil lymphocytes and
about one tenth the rate found in adenoid lymphocytes.
A seasonal effect may contribute to
the large discrepancies found in HBoV
prevalences. Apparently, viruses can
persist in tonsillar tissue well after the
symptomatic phase of illness. In children with no signs of acute respiratory
infection, Drago et al. (7) reported that
45.5% of samples contained viral nucleic acid. Depending on the duration
of persistence, asymptomatic children,
sampled shortly after the season of the
virus in question, would be more likely
to have detectable virus in their tonsillar
tissue. The Longtin et al. study samples
were collected from December through
April; our study samples were collected
from June through September. If HBoV
is seasonal, as has been suggested (3), it
may have been circulating in the target
population before samples were taken
and persisted only in tonsillar tissues.
Thus, if tonsillar tissue from asymptomatic children was obtained within
the persistence period after the HBoV
season, samples would be HBoV positive; those obtained shortly after the
persistence period would have a much
lower rate.
Differences in patient age in the
3 studies may also have contributed
to the different rates observed. The
Longtin et al. group was substantially younger (median age 23 months)
than the Lu et al. group (median age
5 years) or our group (median age 5.9
years). Preliminary seroepidemiology
reports indicate the presence of HBoV
antibodies in >50% of children 2–3
years of age (8,9).
The detection of HBoV in the
tonsillar tissues we tested showed a
higher rate of infection than would be
expected in an asymptomatic population. However, the rate was far lower
than that previously reported for tonsillar tissues (1,4).
1150
Nathalie Clément,1
Gino Battaglioli,
Ryan L. Jensen,2
Bruce C. Schnepp,2
Philip R. Johnson,2
Kirsten St. George,
and R. Michael Linden3
Author affiliations: Mount Sinai School of
Medicine, New York, New York, USA (N.
Clément, R.M. Linden); Wadsworth Center, Albany, New York, USA (G. Battaglioli,
K. St. George); and Nationwide Children’s
Hospital, Columbus, Ohio, USA (R.L. Jensen, B.C. Schnepp, P.R. Johnson)
DOI: 10.3201/eid1507.090102
References
1.
2.
3.
4.
5.
6.
Longtin J, Bastien M, Gilca R, Leblanc
E, de Serres G, Bergeron MG, et al. Human bocavirus infections in hospitalized children and adults. Emerg Infect
Dis. 2008;14:217–21. DOI: 10.3201/
eid1402.070851
Fry AM, Lu X, Chittaganpitch M, Peret
T, Fischer J, Dowell SF, et al. Human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring
hospitalization in Thailand. J Infect Dis.
2007;195:1038–45. DOI: 10.1086/512163
Kesebir D, Vazquez M, Weibel C, Shapiro
ED, Ferguson D, Landry ML, et al. Human bocavirus infection in young children
in the United States: molecular epidemiological profile and clinical characteristics
of a newly emerging respiratory virus.
J Infect Dis. 2006;194:1276–82. DOI:
10.1086/508213
Lu X, Gooding LR, Erdman DD. Human
bocavirus in tonsillar lymphocytes. Emerg
Infect Dis. 2008;14:1332–4. DOI: 10.3201/
eid1408.080300 PMID: 18680679
Chen CL, Jensen RL, Schnepp BC,
Connell MJ, Shell R, Sferra TJ, et al.
Molecular characterization of adenoassociated viruses infecting children. J
Virol. 2005;79:14781–92. DOI: 10.1128/
JVI.79.23.14781-14792.2005
Allander T, Tammi MT, Eriksson M,
Bjerkner A, Tiveljung-Lindell A, Andersson B. Cloning of a human parvovirus
by molecular screening of respiratory
Current affiliation: University of Florida,
Gainesville, Florida, USA.
1
Current affiliation: Children’s Hospital of
Philadelphia, Philadelphia, Pennsylvania,
USA.
2
Current affiliation: King’s College, London,
UK.
3
tract samples. Proc Natl Acad Sci U S A.
2005;102:12891–6. DOI: 10.1073/pnas.
0504666102
7. Drago L, Esposito S, De Vecchi E,
Marchisio P, Blasi F, Baggi E, et al. Detection of respiratory viruses and atypical
bacteria in children’s tonsils and adenoids.
J Clin Microbiol. 2008;46:369–70. DOI:
10.1128/JCM.01819-07
8. Kahn JS, Kesebir D, Cotmore SF,
D’Abramo A, Cosby C, Weibel C, et al.
Seroepidemiology of human bocavirus
defined using recombinant virus-like particles. J Infect Dis. 2008;198:41–50. DOI:
10.1086/588674
9. Lindner J, Karalar L, Zehentmeier S,
Plentz A, Pfister H, Struff W, et al. Humoral immune response against human
bocavirus VP2 virus-like particles. Viral
Immunol. 2008;21:443–9. DOI: 10.1089/
vim.2008.0045
Address for correspondence: Gino Battaglioli,
Laboratory of Viral Diseases, Wadsworth
Center, New York State Department of Health,
Empire State Plaza, PO Box 509, Albany, NY
12201, USA; email: battagli@wadsworth.org
Bartonella
rochalimae and
Other Bartonella
spp. in Fleas, Chile
To the Editor: Fleas are involved
in the natural cycle of different Bartonella spp. Among the 20 currently recognized Bartonella spp., 13 species or
subspecies have been implicated in human disease. Recently, B. rochalimae
was identified in a patient who had received numerous insect bites and subsequently had bacteremia, fever, and
splenomegaly after visiting Peru (1).
A recent study in Taiwan suggested
that rodents could be a reservoir for B.
rochalimae (2), but the vector or other
mechanism of infection remains unknown. We amplified B. rochalimae,
B. clarridgeiae, and B. henselae from
fleas (Pulex irritans and Ctenocephalides felis) collected in Chile and dis-
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 7, July 2009
LETTERS
cuss the role of these fleas as possible
vectors of infection.
From 2005 through 2008, we collected 82 fleas from cats and dogs in
pounds in Chile: 34 P. irritans, 37 C.
felis, and 11 C. canis. Fleas were kept
in 70% ethanol and sent to the Special
Pathogens Laboratory of the Área de
Enfermedades Infecciosas of the Hospital San Pedro, La Rioja, Spain, to be
examined for Bartonella spp. Fleas
were then rinsed in distilled water and
dried on sterile filter paper under a laminar-flow hood. Each flea was crushed
with a sterile pestle, and DNA was
extracted by lysis with 0.7 M ammonium hydroxide. PCR was used to detect Bartonella DNA (according to the
defining criteria for Bartonella spp.);
primers targeted the RNA polymerase
β-subunit–encoding gene (rpoB) and
the citrate synthase gene (gltA) (3–5).
PCR primers for a fragment of the
16/23S rRNA intergenic region and
the heat-shock protein-encoding gene
(groEL) were also used (6,7). Positive
controls (B. henselae strain Marseille,
kindly supplied by Unité des Rickettsies, Faculté de Médecine, Université
de la Méditerranée, Marseille, France)
and negative controls (sterile water
instead of template DNA) were used.
PCR products were purified, and both
strands of each amplicon were subjected to sequence analysis. Nucleotide
sequence homologies were searched
by using BLAST (www.ncbi.nlm.nih.
gov/blast/Blast.cgi).
When rpoB primers were used,
Bartonella spp. were found in 4 C. felis
(4.8%) fleas from cats and in 4 P. irritans (4.8%) fleas from dogs. The same
8 samples were positive when primers for gltA gene and 16/23S rRNA
intergenic region were used. Unfortunately, none of the 82 specimens were
positive when PCR primers targeting
the groEL gene were used. In all experiments, negative controls remained
negative.
Sequencing of the 825-bp rpoB
fragments from the 4 C. felis fleas
indicated that they were most closely
aligned with the gene sequences of B.
clarridgeiae (n = 2, >99% similarity)
and B. henselae (n = 2, 100% similarity). Using gltA (380 bp), we found
100% similarity with B. clarridgeiae
and B. henselae. Accordingly, the
16/23S rRNA amplicons from these
specimens exhibited 100% similarity
with the corresponding sequences of
B. clarridgeiae (154 bp) and B. henselae (172 bp).
Amplicons for the rpoB fragment gene obtained from 4 P. irritans fleas showed highest similarity (97.2%–99.5%) with rpoB of B.
rochalimae. Three were identical, and
we deposited the consensus sequence
in GenBank in 2006 under the name
“uncultured Bartonella sp.” and accession no. DQ858956. The sequence
differed from those described for all
known Bartonella spp. and phylogenetically was most closely related to
B. clarridgeiae (8). The sequence of
the protein encoded by rpoB in these
3 specimens (protein_id ABH09235)
had 3 aa changes (121I→V, 233K→I,
and 274N→E) with respect to the deduced sequence of the RpoB protein
for B. rochalimae. The importance of
these changes remains unknown. The
remaining nucleotide sequence was
recently submitted to GenBank under
accession no. FJ147196, designated
B. rochalimae because isolation of
this new Bartonella spp. was reported
in 2007 (1). These 4 specimens also
yielded positive PCR products for gltA
(380 bp) and 16/23S rRNA (≈175 bp).
Subsequent nucleotide sequence analysis showed 100% homology with the
corresponding partial nucleotide sequences from B. rochalimae.
In 2002, Parola et al. (9) amplified
Bartonella DNA by using PCR with
Pulex spp. fleas collected from persons
in Peru and suggested the existence of
a new Bartonella sp. The nucleotide
sequence of the 16S-23S ribosomal
RNA intergenic spacer obtained from
1 genotype (clone F17688) was nearly
identical to the corresponding sequence of B. rochalimae. This finding
suggests that Pulex spp. fleas could be
vectors.
Cat scratch disease has been
reported in Chile, and B. henselae
has been found in cats in Chile (10).
Thus, our finding of B. henselae and
B. clarridgeiae in C. felis fleas from
Chile confirms the risk for exposure
of humans in contact with cat fleas.
Furthermore, our finding of B. rochalimae in P. irritans fleas from dogs
in Chile supports the possibility that
P. irritans fleas could be vectors for
B. rochalimae. These findings are of
public health importance because they
identify possible vectors of these human pathogens.
Acknowledgments
We are grateful to Lourdes Romero
and Josune García for their contribution to
this work.
Laura Pérez-Martínez,
José M. Venzal,
Daniel González-Acuña,
Aránzazu Portillo,
José R. Blanco,
and José A. Oteo
Author affiliations: Hospital San Pedro-Centro de Investigación Biomédica de La Rioja,
Logroño, Spain (L. Pérez-Martínez, A. Portillo, J.R. Blanco, J.A. Oteo); Universidad de
la República, Montevideo, Uruguay (J.M.
Venzal); and Universidad de Concepción,
Chillán, Chile (D. González-Acuña)
DOI: 10.3201/eid1507.081570
References
1.
Eremeeva ME, Gerns HL, Lydy SL, Goo
JS, Ryan ET, Mathew SS, et al. Bacteremia, fever, and splenomegaly caused by
a newly recognized Bartonella species.
N Engl J Med. 2007;356:2381–7. DOI:
10.1056/NEJMoa065987
2. Lin JW, Chen CY, Cehn WC, Chomel BB,
Chang CC. Isolation of Bartonella species
from rodents in Taiwan including a strain
closely related to ‘Bartonella rochalimae’
from Rattus novergicus. J Med Microbiol. 2008;57:1496–501. DOI: 10.1099/
jmm.0.2008/004671-0
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 7, July 2009
1151
LETTERS
3.
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Renesto P, Gouvernet J, Drancourt M,
Roux V, Raoult D. Use of rpoB gene
analysis for detection and identification of Bartonella species. J Clin Microbiol. 2001;39:430–7. DOI: 10.1128/
JCM.39.2.430-437.2001
Norman AF, Regnery R, Jameson P,
Greene C, Krause DC. Differentiation of
Bartonella-like isolates at the species level
by PCR-restriction fragment length polymorphism in the citrate synthase gene. J
Clin Microbiol. 1995;33:1797–803.
La Scola B, Zeaiter Z, Khamis A, Raoult
D. Gene-sequence–based criteria for
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Jensen WA, Fall M, Rooney J, Kordick
DL, Breitschwerdt EB. Rapid identification and differentiation of Bartonella species using a single-step PCR assay. J Clin
Microbiol. 2000;38:1717–22.
7. Sanogo YO, Zeaiter Z, Caruso G, Merola
F, Shpynov S, Brouqui P, et al. Bartonella
henselae in Ixodes ricinus ticks (Acari:
Ixodida) removed from humans, Belluno Province, Italy. Emerg Infect Dis.
2003;9:329–32.
8. González-Acuña D, Pérez-Martínez L,
Venzal JM, Portillo A, Santibáñez S, Ibarra V, et al. Detection of Bartonella sp. in
Pulex irritans from Chile. In: Abstracts of
the 20th Meeting of the American Society
for Rickettsiology and the 5th International Conference on Bartonella as Emerging
Pathogens; 2006 Sep 2–7; Pacific Grove,
California, USA. Abstract 154.
9.
Parola P, Shpynov S, Montoya M, Lopez
M, Houpikian P, Zeaiter Z, et al. First molecular evidence of new Bartonella spp. in
fleas and a tick from Peru. Am J Trop Med
Hyg. 2002;67:135–6.
10. Ferrés M, Abarca K, Godoy P, García P,
Palavecino E, Méndez G, et al. Presence
of Bartonella henselae in cats: natural
reservoir quantification and human exposition risk of this zoonoses in Chile. Rev
Med Chil. 2005;133:1465–71.
Address for correspondence: José A. Oteo, Área
de Enfermedades Infecciosas, Hospital San
Pedro, C/Piqueras 98-7ª NE, 26006 – Logroño
(La Rioja), Spain; email: jaoteo@riojasalud.es
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 7, July 2009