VECTOR-BORNE AND ZOONOTIC DISEASES

Volume 11, Number 7, 2011

SHORT COMMUNICATION
ª Mary Ann Liebert, Inc.
DOI: 10.1089/vbz.2010.0146

Rickettsia felis and Bartonella henselae

in Fleas from Lebanon

Pamela Angue Mba,1 Jean-Lou Marié,2 Jean-Marc Rolain,1 Bernard Davoust,2

Jean-Claude Beaucournu,3 Didier Raoult,1 and Philippe Parola1

Abstract

A total of 155 fleas collected in 2009 in Lebanon from 16 cats (104 Ctenocephalides felis specimens, 1 C. canis
specimen) and 2 dogs (50 C. canis specimens) were tested for the presence of Rickettsia spp. and Bartonella spp.
using molecular methods, including real-time quantitative polymerase chain reaction (PCR), regular PCR, and
sequencing of amplified PCR products. Rickettsia felis, the agent of the emerging flea-borne spotted fever in
humans, was identified in 17 (16%) C. felis cat fleas. Bartonella henselae, an agent of cat scratch disease, was
identified in three (2.9%) C. felis. Our results emphasize the potential risk of these emerging flea-borne infections
in Lebanon.

Key Words: Bartonella henselae—Fleas—Lebanon—Rickettsia felis.

Introduction DNA was extracted from each specimen (Berrelha et al.

F leas are worldwide vectors of several important zoo-
noses transmitted to humans including plague (Yersinia
pestis) and murine typhus (Rickettsia typhi), and they also play
a role in the cycle of rural epidemic typhus (Rickettsia prowa-
zekii) in the United States (Bitam et al. 2010).
In recent years, fleas have been associated with emerging
human infections worldwide, including flea-borne spotted
fever (also called cat flea typhus) caused by R. felis and the cat
scratch disease (CSD) in connection with Bartonella henselae.
The cat flea, Ctenocephalides felis, has been associated with both
pathogens (Bitam et al. 2010).
Fleas and human flea-borne infections have been rarely
studied in Lebanon (Matossian et al. 1964). In this work, we
aimed to detect Rickettsia spp. and Bartonella spp. in fleas
collected in this country.
Materials and Methods
From September through November 2009, 105 fleas were
collected from 16 cats and 50 fleas were collected from two
dogs at sites in the Naqoura and Dayr Kifa regions in south-
ern Lebanon. They were stored in 70% ethanol, transported
to France, and identified using standard taxonomic keys
(Beaucournu and Launay 1990).
2009). Each sample was tested by real-time quantitative
polymerase chain reaction (qPCR) for the presence of Rick-
ettsia spp. DNA using primers and a Taqman probe targeting
a partial sequence of the citrate synthase gene gltA as well as a
primer/probe set specific for the R. felis chromosomal bioB
gene (Berrelha et al. 2009). Samples that tested positive for
gltA by qPCR but negative in the R. felis-specific qPCR
were tested by regular PCR using primers CS409d and
Rp1258n, which amplify a 396-bp fragment of rickettsial gltA,
and primers 190.70 or 190.180, and 190.701, which amplify a
629- to 632-bp fragment of rickettsial ompA (Berrelha et al.
2009).
All DNA samples were also screened using Bartonella
genus-specific qPCR with a Taqman probe targeting the 16S/
23S rRNA gene intergenic spacer (ITS) (Varagnol et al. 2009).
Samples that tested positive for Bartonella by qPCR were
thereafter tested using standard PCR with primers that am-
plified a variable-size ITS of Bartonella spp. (Parola et al. 2003).
For Bartonella species identification, DNA sequencing reac-
tions were performed for all samples reamplified in standard
Bartonella ITS PCR. Negative controls constituted DNA ex-
tracted from noninfected laboratory ticks. R. felis (qPCR) and
B. elisabethae (ITS PCR) DNA samples served as positive
controls. Sequences were edited, assembled, and identified as
described (Berrelha et al. 2009).

1
Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UMR CNRS 6236-IRD UMR 198, Faculté de Médecine,
WHO Collaborative Center for Rickettsioses and Other Arthropod Borne Bacterial Diseases, Université de la Méditerranée, Marseille, France.
2
Working Group on Animal Epidemiology of the French Forces Health Service, Marseille, France.
3
Laboratoire de Parasitologie et Zoologie Appliquée, Faculté de Médecine, Rennes, France.

991
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Results
With the exception of one flea that was identified as
C. canis, all fleas collected from cats were identified as C. felis
felis. All fleas collected from dogs were identified as C. canis.
All real-time qPCR- and PCR-positive and negative controls
tested as expected. Rickettsia DNA was detected by qPCR in 18
(all C. felis felis) of the 155 specimens; 17 of these 18 Rickettsia-
positive fleas were positive in the R. felis-specific qPCR, and
1 was negative. This one was tested by regular PCR using
primers that amplified parts of the gltA and ompA genes of
rickettsiae. However, after several attempts, no sequence
permitting bacterial identification was obtained.
Bartonella DNA was detected by qPCR in 8 (all C. felis felis)
of the 155 fleas; among them, 5 tested positive by the standard
ITS PCR. Sequencing showed that the amplified 723-bp
product had 100% homology with B. henselae (GenBank
accession number: AJ457177.1) in three of these five fleas. The
quality of the other two sequences obtained was insufficient to
permit molecular identification despite several attempts. One
specimen of C. felis felis was found to be coinfected with both
R. felis and B. henselae.
Discussion
R. felis is an obligate intracellular Gram-negative bacterium
belonging to the spotted fever group of Rickettsia (Pérez-
Osorio et al. 2008). C. felis is currently the only known bio-
logical vector of R. felis, and these fleas are able to maintain
stable infected progeny through transovarial transmission
(Reif and Macaluso 2009). However, molecular evidence of
R. felis has been found in various arthropods (Reif and Ma-
caluso 2009). Flea-borne spotted fever caused by R. felis is to
date only incompletely described (Pérez-Osorio et al. 2008).
The clinical features may include fever, fatigue, headache,
generalized maculopapular rash, inoculation eschar(s), and
lymph nodes. Cases have been reported from Brazil, Mexico,
France, Germany, Spain, Tunisia, Egypt, South Korea, Laos,
and Taiwan (Reif and Macaluso 2009). Recently, a study of 134
patients conducted over 9 months in two Senegalese villages
identified up to 6% of indigenous febrile nonmalaria cases as
R. felis associated (Socolovschi et al. 2010).
B. henselae is a Gram-negative bacterium known as an agent
of CSD. Cats have presented as a reservoir for this bacterium,
with an increasing gradient of the prevalence of infection from
cold climates to warm and humid climates (Chomel and
Kasten 2010). Fleas appear to play an important role in the
maintenance and transmission of B. henselae among cats. The
possibility that B. henselae can be directly transmitted to
humans by the cat flea has not been demonstrated experi-
mentally. Transmission of B. henselae by cat fleas seems to
occur mainly through infected flea feces that is inoculated by
the scratching of the cat’s claws (Chomel and Kasten 2010). In
addition to the cat flea, new potential vectors have been
suggested, including ticks (Chomel and Kasten 2010). CSD is
usually characterized by persistent regional lymphadenopa-
thy. Clinical manifestations may include low-grade fever and
aching, headache, anorexia, and splenomegaly as well as
ANGUE MBA ET AL.
various atypical manifestations (Chomel and Kasten 2010).
B. henselae has been also described as an agent of bacillary
angiomatosis and peliosis in highly immunocompromised
individuals (Chomel and Kasten 2010).
Although we cannot address the precise prevalence or
distribution of R. felis and B. henselae in Lebanon, clinicians
there and elsewhere who may see patients returning from this
country must now be alerted by the presence of these flea-
borne agents in the country.
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Philippe Parola
Unité de Recherche en Maladies Infectieuses
et Tropicales Emergentes (URMITE)
UMR CNRS-IRD 6236-198
Faculté de Médecine
WHO Collaborative Center for Rickettsioses
and Other Arthropod Borne Bacterial Diseases
Université de la Méditerranée
Bd Jean Moulin
13385 Marseille
France
E-mail: philippe.parola@univmed.fr
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