Bartonella species in bat flies (Diptera: Nycteribiidae)
from western Africa
S. A. BILLETER1, D. T. S. HAYMAN2,3,4, A. J. PEEL3,4, K. BAKER3,4, J. L. N. WOOD3,
A. CUNNINGHAM4, R. SUU-IRE5, K. DITTMAR6and M. Y. KOSOY1*
1Centers for Disease Control and Prevention, Division of Vector Borne Diseases, Fort Collins, Colorado, USA
2Rabies and Wildlife Zoonoses Group, Veterinary Laboratories Agency-Weybridge, Surrey, UK
3Cambridge Infectious Diseases Consortium, Department of Veterinary Medicine, University of Cambridge, Cambridge, UK
4Institute of Zoology, Zoological Society of London, London NW1 4RY, UK
5Wildlife Division of the Forestry Commission, Accra, Ghana
6State University of New York at Buffalo, Department of Biological Sciences, Buffalo, New York, USA
(Received 20 July 2011; revised 14 October and 24 October 2011; accepted 27 October 2011; first published online 6 February 2012)
Bat flies are obligate ectoparasites of bats and it has been hypothesized that they may be involved in the transmission of
Bartonella species between bats. A survey was conducted to identify whether Cyclopodia greefi greefi (Diptera:
Nycteribiidae) collected from Ghana and 2 islands in the Gulf of Guinea harbour Bartonella. In total, 137 adult flies
analysis. Bartonella DNA was detected in 91 (66·4%) of the specimens examined and 1 strain of a Bartonella sp., initially
identified in E. helvum blood from Kenya, was obtained from a bat fly collected in Ghana. This is the first study, to our
knowledge, to report the identification and isolation of Bartonella in bat flies from western Africa.
Key words: Bartonella, Cyclopodia bat fly, Eidolon helvum, bat, Africa, PCR, culture.
Bartonella species are gram-negative bacteria that
infect erythrocytes of their respective hosts and most
are believed to be transmitted by arthropod vectors
(Billeter et al. 2008). There are currently more
than 20 recognized Bartonella species and many of
those are known or suspected zoonotic pathogens
(Breitschwerdt et al. 2010). Bartonella DNA has also
been detected in a wide variety of biting arthropods
including ticks, fleas, lice, mites, flies, and keds
(Billeter et al. 2008). The detection of DNA,
however, does not demonstrate viability of the
organism or vector competency of the arthropod.
To date, only 6 Bartonella species have been
confirmed to be transmitted by an arthropod exper-
verrucarum sandflies, B. quintana is transmitted by
Pediculus humanus body lice, B. birtlesii is trans-
mitted by Ixodes ricinus ticks, B. henselae is trans-
mitted by Ctenocephalides felis fleas, and B. grahamii
and B. taylorii are transmitted by Ctenophthalmus
nobilis fleas (Swift, 1920; Battistini, 1931; Chomel
et al. 1996; Bown et al. 2004; Billeter et al. 2008; Reis
et al. 2011).
Eidolon helvum, the straw-coloured fruit bat, is
distributed throughout much of sub-Saharan Africa
and feeds on flowers, leaves, and fruits. These bats
tend to roost in colonies of over 100 000, often in
urban areas, and are used as a human food source in
Central and West Africa. Bats are known reservoirs
of zoonotic viruses including rabies, Nipah virus,
coronaviruses, and others, but their role in the
transmission of human pathogenic bacteria has not
been explored in detail (Wong et al. 2007). Results
from our laboratory have shown that some bats,
including E. helvum, are carriers for several unique
Bartonella species but their ability to transmit these
organisms to humans and other animals is unknown
(Kosoy et al. 2010). It is also unknown whether any
arthropod vectors are responsible for the trans-
mission of Bartonella species between bats.
Bat flies (Diptera) are obligate, blood-sucking
ectoparasites often found living in the fur and on
wing membranes of the bat host (Dick and Patterson,
2006). Bat flies are most closely related to tse-tse flies
(Glossinidae) and Hippoboscid flies and all belong to
the same super family, Hippoboscoidea (Dittmar
et al. 2006). Bat flies are divided into 2 families:
Streblidae, most commonly found in the Western
Hemisphere, and Nycteribiidae, predominantly in
the Eastern Hemisphere (Dick and Patterson, 2006).
Although Bartonella DNA was detected in a bat
fly, Trichobius major (Diptera: Streblidae) collected
from a Myotis austroriparius in Florida (Reeves et al.
* Corresponding author: Centers for Disease Control and
Prevention, Department of Vector Borne Diseases, 3150
Rampart Road, Fort Collins, CO 80521, USA. Tel:
+9702663522. Fax: +970 2254257. E-mail: mck3@cdc.
Parasitology (2012), 139, 324–329.
© Cambridge University Press 2012
2005), no surveys have been performed to screen
Nycteribiidae flies for the presence of Bartonella or
demonstrate viability of the organism in these flies.
Because of this, a study was performed to examine
bat flies from western Africa for the presence of
Bartonella using culture and PCR analysis.
MATERIALS AND METHODS
Bat fly collection and identification
Bat flies were collected from E. helvum in Ghana and
on 2 islands in the Gulf of Guinea, Annobón and
Bioko; in total 137 flies were examined (Fig. 1). From
the study site in Accra, Ghana, 46 adult flies were
collected, placed into pots, and transferred to −80 °C
shortly thereafter. From the island of Annobón, a
total of 32 flies was collected: 16 were suspended in
70% ethanol and 16 were placed at −80 °C. A total of
59 bat flies was collected from E. helvum on the
island of Bioko and all samples were placed in tubes
containing 70% ethanol. Samples were subsequently
shipped overnight on dry ice (those samples frozen at
−80 °C) or ice packs (samples suspended in ethanol)
to the Ft. Collins CDC, Bartonella Laboratory.
Upon arrival, all samples were placed at −80 °C
until further analysis. After DNA extraction, exo-
skeletons were kept as DNA vouchers and were
subsequently slide mounted in PVA (BioQuip
Products, Rancho Dominguez, CA, USA). Bat flies
were sexed and identified using the species keys of
Theodor (1967). All specimens keyed to Cyclopodia
greefi greefi, based on the characteristic setation of the
fore-tibiae, the presence of a quadratic patch of long
dorsal abdominal setae on the female, characteristic
head morphology,abroad,openhalteregroove,and a
single dorsal thoracic seta. Specimens collected from
Annobón were slightly heavier sclerotized, but
otherwise adhered to the key.
Culturing of Bartonella spp. from flies
All frozen bat fly specimens were thawed, subjected
to surface sterilization using a Wescodyne wash
(20 min) followed by a 70% ethanol wash (5min),
and were then rinsed in sterile 1x PBS (0·15 M, pH:
7·50, Atlanta, CDC). Flies were cut in half long-
itudinally using a sterile scalpel blade so that half
analysis.Forculture,fliesweresuspended in500 μlof
Brain Heart Infusion (BHI) broth (CDC) containing
20% fungizone; 300 μl was aliquoted onto a BHI agar
plate containing 5 or 10% defibrinated rabbit blood
(CDC). Plates were placed at 35 °C, 5% CO2and
colonies were subcultured by streaking onto a fresh
rabbit blood agar plate.
DNA extraction and PCR amplification
DNA extraction of 137 adult bat flies and second
passage colonies was performed using a Qiagen
Fig. 1. Map of western Africa, including the sites of bat fly collection. A total of 137 adult Cyclopodia greefi greefi flies
were collected from Eidolon helvum: 46 flies from Accra, Ghana, 32 flies from the island of Annobón, and 59 flies from
the island of Bioko.
325Bartonella species in African bat flies
QIAamp tissue kit (QIAGEN, Valencia, CA, USA)
according to the manufacturer’s instructions. Prior
to DNA extraction, the bat flies in ethanol were
triturated using a sterile scalpel blade. For the
cultured Bartonella, 6 individual colonies from 1
isolatewere picked using a sterile loop and suspended
in 200 μl of BHI broth. Bat flies were examined for
the presence of Bartonella DNA by conventional
PCR. Primers 443f (5′ GCT ATG TCT GCA TTC
TAT CA) (Birtles and Raoult, 1996) and 1210r
(5′ GAT CYT CAA TCA TTT CTT TCC A) were
designed to amplify a portion of the citrate synthase
gene, gltA. Each 50 μl PCR reaction contained
28·75 μl of nuclease-free water, 5x PCR buffer
containing MgCl2, 0·2mM dNTPs, 15 pmol of
each primer, and 1·25 U of GoTaq DNA polymerase
(Promega, Madison, WI, USA). PCR wasperformed
using a Bio-Rad Laboratories iCycler (Hercules, CA,
USA) with the following cycling conditions: 94 °C
for 2 min followed by 45 cycles of 94°C for 30sec,
48 °C for 1 min, 72 °C for 1min, and 1 cycle of 72 °C
for 7 min.
PCR products were separated using electro-
phoresis in a 1·5% agarose gel containing ethidium
bromide and were visualized under UV light.
The positivecontrol consisted
DNA and nuclease-free water was used as a negative
Gene sequencing and analysis
Amplicons were purified using the QIAquick PCR
purification kit (QIAGEN) and sequenced using an
Applied Biosystems Model 3130 Genetic Analyzer
(Applied Biosystems, Foster City, CA, USA). DNA
sequences were analysed using the Lasergene v8
sequence analysis software (DNASTAR, Madison,
WI, USA) to determine a consensus sequence for the
PCR product from each bat fly. All sequences from
this study were subsequently cropped, *307 bp, for
further phylogenetic analysis. Sequences obtained in
this study were considered similar to validated
Bartonella species if similarity over the gltA fragment
was >96·0% (La Scola et al. 2003). The Clustal W
program in Megalign (Lasergene) was used to
compare sequences obtained from this study to
Bartonella sequences available in GenBank. GltA
sequences from 5 novel Bartonella species, isolated
from E. helvum in Kenya, were also included in
the phylogenetic analyses. Five species, identified
HM363765–HM363768) and ‘E-124’ (GenBank
The neighbor-joining (N-J) method based on
the Kimura 2-parameter model and the boot-
strap calculations were carried out with 1000 re-
Nucleotide sequence Accession numbers
Sequences, representative of each genotype obtained
from this study, were deposited in GenBank under
Accession numbers JN172035 –JN172074.
Prevalence of Bartonella DNA in bat flies
Of the 137 adult bat flies examined, 66·4% were PCR
positive for the presence of Bartonella DNA using
gltA specific primers (Table 1). Positivity from each
location ranged from 56·5% in Ghana to 71·9% on the
island of Annobón.
Of 82 sequences examined, a total of 39 genotypes
(genotype consisting of 1 or more nucleotide differ-
ences) were found (Table 2). Eight genotypes were
represented by 2–30 identical sequences. The
sequence similarity among Bartonella sequences
detected in bat flies ranged between 71·4 and 100%,
while similarity among Bartonella sequences ob-
tained from bat flies and those identified previously
from E. helvum ranged from 71·9 to 100%. Fifty-
five of the 82 sequences were 596% similar to
Bartonella previously isolated from E. helvum
or other Bartonella species, suggesting that the
remaining 27 sequences may represent novel species
Isolation of a Bartonella sp. from a bat fly
Although attempts to culture Bartonella spp. from
bat flies were largely unsuccessful due to overgrowth
of contaminants, a single isolate from a bat fly was
obtained. A Bartonella sp. isolate, 98·4% identical
to Bartonella sp. ‘E1-105’ isolated from bats in
Kenya based on gltA sequence analysis (GenBank
Accession: HM363765), was cultured from 1 bat fly
collected in Ghana (fly Cg 23 Q22-1). Further
molecular characterization was performed by se-
quence analysis of the 16S rRNA gene, ftsZ,
and rpoB using methods described previously
Table 1. Prevalence of Bartonella DNA in adult
bat flies from western Africa
Total number of
adult bat flies
% PCR positive
S. A. Billeter and others
(Marchesi et al. 1998; Renesto et al. 2001; Zeaiter
et al. 2002). Comparison of sequence data from
Bartonella ‘E1-105’ demonstrated sequence simi-
larity of 100%, 100%, and 99·8% within the 16S
rRNA gene, ftsZ, and rpoB genes, respectively
‘E1-105’: HM363785, HM363770 and HM363775).
Within the current study, we demonstrated (1) the
presence of Bartonella DNA in bat flies from western
Africa and (2) the successful culture of Bartonella
from a bat fly collected in Ghana. Bartonella
DNA has previously been detected in bat ectopar-
asites, specifically in a T. major bat fly, Cimex
adjunctus (Hemiptera: Cimicidae) bat bug, Carios
texanus (Siphonaptera: Ischnopsyllidae) flea, and a
Steatonyssus sp. mite (Acari: Mesostigmata) (Loftis
et al. 2005; Reeves et al. 2005, 2006a, 2007). Our
data, unlike previous surveys, reveals that a large
percentage (up to 66·4%) of C. greefi greefi harboured
Bartonella DNAand,inatleast1 case,viabilityofthe
bacteria could be confirmed. More than half (65·9%
of 82) of the sequences obtained were identical or
similar to Bartonella species previously isolated from
E. helvum (Kosoy et al. 2010), suggesting a potential
role of bat flies in the transmission of Bartonella
between bats. When compared with Bartonella
species isolated from other bat species in Kenya, the
bat fly Bartonella genotypes detected in this study
appear to be very host specific (data not shown).
Interestingly, 27 (32·9%) Bartonella sequences did
appear to be unique to bat flies. It is unclear, at this
point, whether Bartonella species may circulate
among bat flies outside of the host or whether these
27 sequences represent potential symbionts.
Previous investigations have also pointed to the
likely potential of Bartonella transmission by other
hippoboscid flies. Lipoptena cervi, or the deer ked, is
the suspected vector of B. schoenbuchensis in deer.
Dehio et al. (2004) successfully isolated B. schoenbu-
and demonstrated the presence of the bacteria in the
mid-gut of the flies. Bartonella DNA has also been
detected in L. cervi from Massachusetts and
L. mazamae collected in South Carolina (Reeves
et al. 2006b; Matsumoto et al. 2008). Halos et al.
of L. cervi (94% of 48 adults), Hippobosca equina (71%
of 17 adults), and Melophagus ovinus (100% of 20
adults) collected from 4 different ruminant species.
Bartonella DNA was also found in 100% (10 total) of
the M. ovinus pupae screened suggesting the bacteria
may be vertically transmitted in this species.
Further investigation is warranted to decipher
what role, if any, bat flies or other bat ectoparasites
might play in the transmission of Bartonella spp.
between bats. Future studies will be performed to
screen bat flies from other bat species and locations
for the presence of Bartonella. Attempts will also be
made to isolate viable Bartonella from pupae to
may occur, as suggested by Halos et al. (2004). Bat
flies undergo adenotrophic vivaparity (i.e. the com-
plete development of 3 larval stages inside the adult
female) and deposition of a single pre-pupae. This
unique reproductive ability may facilitate vertical
transmission of parasites, including Bartonella spp.
going to determine whether these agents are respon-
sible for human illnesses.
Table 2. The total number of 39 Bartonella genotypes, from 82 sequences examined, detected in bat flies
from western Africa
(Genotypes with identical sequences were given identification numbers of 1–8. All remaining
genotypes were unique.)
Total number of sequences detected per genotype
(representative GenBank Accession number)
from each location
327 Bartonella species in African bat flies
S.A. Billeter is supported through the American Society of
Microbiology/Centers for Disease Control and Prevention
Post-Doctoral Associates Program in Infectious Diseases
and Public Health Microbiology. D. T. S. Hayman
is supported by a Wellcome Trust Research Training
Fellowship, J. L. N. Wood is supported by the Alborado
Trust, and both are supported by the RAPIDD program of
the Science & Technology, Directorate, Department of
Fig. 2. Tree topology displaying similarity of Bartonella DNA detected in Cyclopodia greefi greefi with known
Bartonella sequences based upon partial citrate synthase gene, gltA. The topology was constructed by the
neighbor-joining method based on the Kimura-2 parameter model of nucleotide substitution. Bootstrap values are based
on 1000 replicates. The tree was rooted by the use of Brucella melitensis 16MTas the out-group. Bartonella sequences
obtained from 5 Eidolon helvum, collected in Kenya (Kosoy et al. 2010), are included in the tree (GenBank Accession
numbers: HM363765 –HM363768 and JN190887). Unique Bartonella sequences identified in bat flies from Annobón
are represented by GenBank Accession numbers JN172049–JN172057, Bartonella sequences obtained from bat flies
from Bioko are represented by GenBank Accession numbers JN172058 –JN172074, and Bartonella sequences obtained
from Ghana bat flies are represented by GenBank Accession numbers JN172035 –JN172048.
S. A. Billeter and others
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329Bartonella species in African bat flies