Am. J. Trop. Med. Hyg., 92(3), 2015, pp. 617–619
doi:10.4269/ajtmh.14-0424
Copyright © 2015 by The American Society of Tropical Medicine and Hygiene
Rickettsia and Bartonella Species in Fleas from Reunion Island
Constentin Dieme, Philippe Parola, Vanina Guernier, Erwan Lagadec, Gildas Le Minter, Elsa Balleydier, Frederic Pagès,
Koussay Dellagi, Pablo Tortosa, Didier Raoult, and Cristina Socolovschi*
Aix Marseille Université, URMITE, Marseille, WHO Coll. Centre for Rickettsioses and Other Arthropod Borne Bacterial Diseases, France;
Centre de Recherche et de Veille sur les Maladies Emergentes dans l’Océan Indien, Plateforme de Recherche CYROI, Sainte Clotilde, Reunion
Island, France; Institut de Recherche pour le Développement, Reunion Island, France; Regional Office of the French Institute for Public Health
Surveillance (Cire OI - Institut de Veille Sanitaire), Saint Denis, Reunion Island, France; Université de La Reunion,
Joint Chair CNRS-Université de La Reunion, Sainte Clotilde, Reunion Island, France
Abstract. Rickettsia felis, Rickettsia typhi, and Bartonella DNA was detected by molecular tools in 12% of Rattus
rattus fleas (Xenopsylla species) collected from Reunion Island. One-third of the infested commensal rodents captured
during 1 year carried at least one infected flea. As clinical signs of these zoonoses are non-specific, they are
often misdiagnosed.
bution and ecology of the collected fleas and their animal hosts
are the subject of another study.7 A total of 205 flea DNA
samples extracted as previously described,8 were sent in dry
ice to the World Health Organization (WHO) Center for
Rickettsial Diseases, Marseille. These fleas were collected from
59 small mammals including 52 R. rattus, two R. norvegicus, four
S. murinus, and one M. musculus. We analyzed four flea species,
i.e., Xenopsylla cheopis (134/205), Xenopsylla brasiliensis
(57/205), Leptopsylla segnis (13/205), and Echidnophaga
gallinacea (1/205), for the presence of Rickettsia and Bartonella
DNA by quantitative polymerase chain reaction (qPCR)
using a CFX96qPCR Detection System (Bio-Rad, Marnes-la
Coquette, France). All positive (R. felis, R. typhi, and Bartonella
elizabethae DNA) and negative (qPCR mix and DNA extracted
from laboratory free bacteria fleas) controls used in the qPCR
and standard PCR assays showed the expected results.
Rickettsia felis DNA was assessed using primers:
Rfel_phosp_MBF, 5 ¢-GCAAACATCGGTGAAATTGA-3 ¢,
and Rfel_phosp_MBR, 5 ¢-GCCACTGTGCTTCACAAACA3 ¢, and the probe Rfel_phosp_MBP, 6FAM-CCGCTTCGTT
ATCCGTGGGACC, targeting the phosphatase gene. Positive
results were confirmed by a second qPCR assay targeting the
guanosine polyphosphate gene using the primers Rfel_ guano_
MBF, 5 ¢GCATATACTTTATTGTGCGCAAGTT-3 ¢, and
Rfel_ guano_MBR, 5 ¢-TTTATCGATTGACAGAAGAAGA
AATCA-3 ¢, and probe Rfel_ guano_MBP, 6FAM-TCGCT
TTTTGGGATTGTTTGCCAGA. We screened the DNA
samples by qPCR for typhus-group rickettsiae with a Rickettsiaspecific glycosyltransferase gene-based Rpr331 system.9 Positive samples were further confirmed with amplification of
the Rpr 274P gene.4 Samples were considered positive when
two amplifications were obtained targeting two different specific genes. Subsequently, DNA samples were screened using
Bartonella genus-specific qPCR with a Taqman probe targeting
the 16S/23S rRNA gene intergenic spacer (ITS).10
Bacterial DNA was detected in 10.73% (22 of 205) of
the fleas by qPCR, including X. cheopis (12%, 16 of 134) and
X. brasiliensis fleas (10.5%, 6 of 57) collected from 15
R. rattus of 59 infested small mammals (25%). Rickettsia felis
was detected in 5 of 205 (2.44%) flea specimens, including
four X. cheopis and one X. brasiliensis collected from three
different R. rattus individuals. Rickettsia typhi was detected in
three (1.46%) X. cheopis fleas collected from three different
R. rattus individuals. Bartonella DNA was detected by qPCR
in 14 (6.83%) flea specimens, including nine X. cheopis
and five X. brasiliensis collected from 11 different R. rattus
INTRODUCTION
Adult male and female fleas are obligate hematophagous
ectoparasites of mammals and birds throughout the world and
can contaminate their hosts with bacteria, viruses, and bloodborne parasites.1 The two most common routes of pathogen
transmission by fleas are 1) oral route, by the regurgitation of
blood meals at the flea-bite site; and 2) fecal, though skin lesions
contaminated with infected fecal pellets by scratching.1 Fleas
are vectors of several important bacterial zoonoses, including
plague (Yersinia pestis), bartonelloses (several Bartonella species), and rickettsioses such as murine typhus (Rickettsia
typhi), flea-borne spotted fever (Rickettsia felis), and occasionally sylvatic epidemic typhus (Rickettsia prowazekii).1,2
Reunion is a tropical oceanic island of volcanic origin
located in the Indian Ocean, East of Madagascar. To date, no
information is available on the distribution of flea species and
flea-borne zoonoses on this island. Several murine typhus
cases were recently confirmed using serological and molecular tools in travelers and autochthonous people from Reunion
Island.3,4 In addition, the seroprevalence rate of bartonellosis
in dogs was estimated to be ~10%.5 The aim of this study was
to analyze the presence of Rickettsia and Bartonella species
in fleas sampled from small mammals on this island, which
has favorable climatic and ecologic conditions for the proliferation of fleas and their hosts.
THE STUDY
+
+
During a 1-year survey (2012–2013), fleas were collected
from small terrestrial mammals, including the black rat (Rattus
rattus), the brown rat (Rattus norvegicus), the Asian house
shrew (Suncus murinus), the house mouse (Mus musculus),
and the tailless tenrec (Tenrec ecaudatus), captured at 19 localities (Figure 1) on Reunion Island in various biotopes. Wire
cage live traps (29 18 12 cm) were used for rat and tenrec
trapping, and Sherman live traps were used for mice and
shrews. All animal procedures carried out in this study were
approved by the French Institutional Ethical Committee
(CYROI) under no. 114. Fleas were manually collected with a
brush or forceps and identified to the species level using the
morphological criteria.6 The detailed descriptions of the distri*Address correspondence to Cristina Scolovschi, URMITE, Faculté
de Médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5 France.
E-mail: cr_socolovschi@yahoo.com
617
618
DIEME AND OTHERS
Figure 1. Map of risk for Rickettsia and Bartonella species on Reunion Island. Red stars: the localities (Port: 20 °55 ¢S, 55 °19E, Sans Souci:
21 °01 ¢S, 55 °30 ¢E, Trois Bassin: 21 °06 ¢S, 55 °17 ¢E, Saint Leu: 21 °10 ¢S, 55 °17 ¢E) where fleas were found infected for Rickettsia and Bartonella
species. Blue stars: the localities where no fleas or non-infected fleas were collected on captured mammals.
individuals. Among these positive samples, two samples
tested positive by standard PCR targeting the 972-bp ITS
fragment.10 Sequence analyses using CHROMAS-PRO version 1.5 showed that one sequence harbored 99.56% (691 of
694) similarity with Bartonella queenslandensis (GenBank
accession no.: EU111800); the second had 99.89% (971 of
972) homology with Bartonella sp. 1.1C (GenBank accession
no.: FN645496) from a R. norvegicus isolated from Taichung,
Taiwan.11 The geographical distribution of the infected fleas
is shown in Figure 1 and Table 1.
CONCLUSION
In this study, R. felis, R. typhi, and Bartonella spp., including Bartonella queenslandensis, and Bartonella sp. 1.1C, were
detected using molecular tools in Xenopsylla fleas collected
from R. rattus on Reunion Island. Almost one-third of the
infested rats (15 of 54) carried at least one infected
Xenopsylla flea. Bartonella species are zoonotic facultative
intracellular parasites of both wild and domestic animals, and
more than 20 species have been described.12 The pathogenicity of B. queenslandensis, which has been isolated from small
mammals from several Asian countries and detected in
X. cheopis fleas, is unknown.13 The analysis of the genome of
Bartonella sp. 1.1C, isolated from R. norvegicus, revealed that
this species belongs to lineage 3, which contains some zoonotic pathogens.12 Unfortunately, the Bartonella DNA load
that was detected using qPCR was low, and we failed to
amplify and sequence the standard PCR product. Further
study is needed to test the tissues of these small animals for
the existence of other Bartonella species. Ten percent (95 if
960) of the captured mammals were infested with fleas. As
Table 1
Detection of Rickettsia and Bartonella species in fleas, Reunion Island
No. of
tested fleas
No. (%) of
positive fleas
Rickettsia spp.
(no. of infected fleas, localities)
Bartonella spp. (no. of infected fleas, localities)
Xenopsylla cheopis
134
16 (11.94)
Xenopsylla brasiliensis
Leptopsylla segnis
Echidnophaga gallinacea
Total
57
13
1
205
6 (10.52)
–
–
22 (10.73)
R. felis (4, St. Leu)
R. typhi (3, Trois Bassin - 1;
Port - 2)
R. felis (1, Sans Souci)
Bartonella spp. (7: Port - 2, St; Leu - 4; Trois Bassin - 1)
B. queenslandensis (1, St. Leu)
Bartonella spp. 1-1C (1, St. Leu)
Bartonella spp. (5, Sans Souci)
8 (3.90)
14 (6.83)
Flea species
RICKETTSIA AND BARTONELLA SPECIES IN FLEAS FROM REUNION ISLAND
Xenopsylla fleas are competent vectors, these Bartonella species could be incidentally transmitted to other hosts such as
humans on Reunion Island.
Rickettsia species are obligate gram-negative intracellular
bacteria vectorized only by hematophagous arthropods.1 Rickettsia felis is an emergent pathogen belonging to the Spotted
Fever Group Rickettsia, with a worldwide distribution.2 It has
previously been detected in several non- and hematophagous
ectoparasites, including Xenopsylla fleas14; however, the only
known biological vector is the cat flea Ctenocephalides felis.2
The identification of R. typhi in 2% of X. cheopis (Oriental
rat flea) collected from the commensal rat R. rattus, including
in the neighborhood of murine typhus cases, illustrates the
life cycle of this pathogen (rat-flea-rat) on this island.
Xenopsylla cheopis remains infectious throughout its life,
from 10 days to a year after an infected blood meal.15 Experimental and field studies have shown that X. cheopis is the
main vector of murine typhus.15 The clinical signs of murine
typhus and R. felis infection are quite similar: high fever,
headache, weakness, generalized pain, and sometimes a generalized rash.2,4 Murine typhus has been diagnosed in recent
years on Reunion Island,3,4 but R. felis infection has not, even
though 15% of febrile patients in Senegal, with the same
climate conditions, tested positive for the latter.16 In addition,
the previous group of symptoms is similar to those of a range
of other bacterial and viral infectious diseases.4 Recently, a
Chikungunya infection study in Reunion Island reported that
30% of patients recruited on the basis of clinical presentation
with acute febrile arthralgia during an epidemic period were
excluded from the diagnosis of the viral infection, which may
reflect that many cases of other infections such as rickettsioses
may be responsible for these symptoms, including severe ones,
which are likely misdiagnosed.17 Within this context, preventive measures should rely on arthropod surveillance and minimizing the risk of exposure in areas of endemicity.
Received July 8, 2014. Accepted for publication September 19, 2014.
Published online February 2, 2015.
Acknowledgments: We thank the Direction de l’Environnement,
de l’Aménagement et du Logement (DEAL Reunion) for issuing
permits and the Office National des Forêts (ONF) and the Parc
National de La Reunion who gave authorizations for the trapping of
Tenrec eucaudatus within protected areas (DIR/I/2013/008).
Financial support: Sampling was supported by funding from CRVOI,
ERDF/French Government/Regional Council of Reunion Island,
FEDER POCT “LeptOI”, no. 32913.
Disclaimer: The authors declare no conflicts of interest.
Authors’ addresses: Constentin Dieme, Philippe Parola, Didier Raoult,
and Cristina Socolovschi, Aix Marseille University, URMITE,
Marseille, WHO Coll. Centre for Rickettsioses and Other Arthropod
Borne Bacterial Diseases, Marseille, France, E-mails: caiusdieme@
yahoo.fr, philippe.parola@univ-amu.fr, didier.raoult@gmail.com, and
cr_socolovschi@yahoo.com. Vanina Guernier, Erwan Lagadec, and
Pablo Tortosa, Centre de Recherche et de Veille sur les Maladies
Emergentes dans l’Océan Indien, Plateforme de Recherche CYROI,
Sainte Clotilde, Réunion, E-mails: vanina.guernier@ird.fr, erwan
.lagadec69@yahoo.fr, and pablo.tortosa@ird.fr. Gildas Le Minter,
Institut de Recherche pour le Développement, Sainte Clotilde,
Réunion, E-mail: leminterbzh@yahoo.fr. Elsa Balleydier and Frederic
Pagès, Regional office of the French Institute for Public Health Surveillance (Cire OI - Institut de Veille Sanitaire), Saint Denis, Réunion,
E-mail: elsa.balleydier@ars.sante.fr and frederic.pages@ars.sante.fr.
Koussay Dellagi, Centre de Recherche et de Veille sur les Maladies
619
Émergentes dans l’Océan Indien (CRVOI), Virology Unit, Saint
Denis, Réunion, Institut de Recherche pour le Développement
(IRD), Saint Denis, Réunion, E-mail: koussay.dellagi@ird.fr.
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