Acta Tropica 150 (2015) 166–170
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Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica
Natural infection of bats with Leishmania in Ethiopia
Aysheshm Kassahun a,∗ , Jovana Sadlova a , Petr Benda b,c , Tatiana Kostalova a ,
Alon Warburg d , Asrat Hailu e , Gad Baneth f , Petr Volf a , Jan Votypka a
a
Department of Parasitology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 44 Prague 2, Czech Republic
Department of Zoology, National Museum (Natural History), Vaclavske nam. 68, 115 79 Prague 1, Czech Republic
Department of Zoology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 44 Prague 2, Czech Republic
d
Department of Microbiology and Molecular Genetics, The Institute for Medical Research Israel-Canada, The Kuvin Centre for the Study of Infectious and
Tropical Diseases, Hadassah Medical School, The Hebrew University of Jerusalem, Jerusalem 91120, Israel
e
Department of Microbiology, Immunology & Parasitology, Faculty of Medicine, Addis Ababa University, P.O. Box 9086, Addis Ababa, Ethiopia
f
School of Veterinary Medicine, Hebrew University, P.O. Box 12, Rehovot 76100, Israel
b
c
a r t i c l e
i n f o
Article history:
Received 12 May 2015
Received in revised form 24 July 2015
Accepted 27 July 2015
Available online 29 July 2015
Keywords:
Bats
Natural infection
kDNA
ITS1
a b s t r a c t
The leishmaniases, a group of diseases with a worldwide-distribution, are caused by different species of
Leishmania parasites. Both cutaneous and visceral leishmaniasis remain important public health problems
in Ethiopia. Epidemiological cycles of these protozoans involve various sand fly (Diptera: Psychodidae)
vectors and mammalian hosts, including humans. In recent years, Leishmania infections in bats have
been reported in the New World countries endemic to leishmaniasis. The aim of this study was to survey
natural Leishmania infection in bats collected from various regions of Ethiopia. Total DNA was isolated
from spleens of 163 bats belonging to 23 species and 18 genera. Leishmania infection was detected by
real-time (RT) PCR targeting a kinetoplast (k) DNA and internal transcribed spacer one (ITS1) gene of the
parasite. Detection was confirmed by sequencing of the PCR products. Leishmania kDNA was detected
in eight (4.9%) bats; four of them had been captured in the Aba-Roba and Awash-Methara regions that
are endemic for leishmaniasis, while the other four specimens originated from non-endemic localities of
Metu, Bedele and Masha. Leishmania isolates from two bats were confirmed by ITS1 PCR to be Leishmania
tropica and Leishmania major, isolated from two individual bats, Cardioderma cor and Nycteris hispida,
respectively. These results represent the first confirmed observation of natural infection of bats with
the Old World Leishmania. Hence, bats should be considered putative hosts of Leishmania spp. affecting
humans with a significant role in the transmission
© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
In Ethiopia, leishmaniases, caused by protozoan parasites of the
genus Leishmania and transmitted by the bite of female sand flies,
are diseases of significant public health importance. The country
is endemic for two human disease presentations: cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL, kala-azar). Cutaneous
leishmaniasis is widely distributed and usually prevalent in highland areas with occasional reports in the lowland regions of Omo
(south) and Awash (central east) (Hailu et al., 2006a). The annual
∗ Corresponding author.
E-mail addresses: ayshek2000@yahoo.com (A. Kassahun), jovanas@seznam.cz
(J. Sadlova), petr benda@nm.cz (P. Benda), tatianakostalova@gmail.com
(T. Kostalova), alonw@ekmd.huji.ac.il (A. Warburg), hailu a2004@yahoo.com
(A. Hailu), gad.baneth@mail.huji.ac.il (G. Baneth), volf@cesnet.cz (P. Volf),
jan.votypka@natur.cuni.cz (J. Votypka).
incidence of CL ranges from 20,000 to 50,000 cases, but this is probably an under-estimate (Alvar et al., 2012), with over 28 million
people residing in regions with risk of transmission (Seid et al.,
2014). The main causative agent of CL in Ethiopia is Leishmania
aethiopica, however, infections due to Leishmania tropica and Leishmania major were also reported in the country (Hailu et al., 2006a,b;
Abbasi et al., 2013). Visceral leishmaniasis affecting up to 7400 people annually in the country is the most severe form and is fatal, if
left untreated. The VL foci lie in the south-west lowland savannah
and the north-west semi-arid plains of the country with sporadic
cases in highland areas of the Libo Kemkem district (north), the
Awash valley (center) and further in the east of the country, bordering Kenya and Somalia (Leta et al., 2014; Hailu et al., 2006a).
The causative agent of human VL in Ethiopia is Leishmania donovani
(Hailu et al., 2006a).
Cutaneous leishmaniasis caused by L. aethiopica and L. major
is commonly zoonotic (Ashford et al., 1973; 2000; Lemma et al.,
2009 Lemma et al., 2009). Although being the agent of anthro-
http://dx.doi.org/10.1016/j.actatropica.2015.07.024
0001-706X/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.
0/).
A. Kassahun et al. / Acta Tropica 150 (2015) 166–170
ponotic CL in urban endemic settings, L. tropica has been strongly
suspected to be zoonotic in some foci (Sang et al., 1994; Jacobson,
2003; Svobodova et al., 2003). In Ethiopia, rock hyraxes were found
infected with L. aethiopica, suggesting a zoonotic cycle of the parasite (Ashford et al., 1973; Lemma et al., 2009). Recently, L. tropica
DNA was detected in spleens of rodents in areas where human cases
have been reported (Kassahun et al., 2015). However, no study in
Ethiopia demonstrated natural infection in animals by L. major.
Most reports agree that like the Indian sub-continent, VL in
East Africa is assumed to be anthroponotic (Chappuis et al., 2007).
Nevertheless, there is evidence for the possible involvement of
zoonotic transmission with uncertain reservoir hosts (Ashford,
2000). Recently, natural infections of dogs (Bashaye et al., 2009),
domestic animals (Rohousova et al., 2015) and rodents (Kassahun
et al., 2015) with L. donovani complex were reported in Ethiopia.
Natural infections by various Leishmania species have been
repeatedly reported in domestic, peridomestic and wild animals,
which dogs and rodents being the most commonly investigated
animals and traditionally considered reservoirs (Baneth and Aroch,
2008). However, recent investigations of Leishmania parasites in
animals including hares (Jimenez et al., 2013), and marsupials
(Roque and Jansen, 2014) have diverted attention to other possible
sylvatic reservoir hosts in endemic leishmaniasis foci.
Bats ecology and innate behavioral details highlight their prime
importance in the reservoir system of infectious diseases such as
Ebola virus (Leroy et al., 2005) and various kinetoplastids transmitted by vectors (Lord and Brooks, 2014). Bats were also suggested as
possible natural blood source for sand flies after laboratory feeding procedure (Lampo et al., 2000) and known to host several
trypanosomes transmitted by sand flies (McConnell and Correa
1964; Williams, 1976). Importantly, being cave-dwelling organisms, bats and sand flies frequently share living habitats where
ample opportunity exists for sand flies to feed on bats (Feliciangeli,
2004). Natural Leishmania infection in bats has been reported in
New World leishmaniasis foci and the findings suggested their possible epidemiological involvement in the transmission cycle (Lima
et al., 2008; Savani et al., 2010; Shapiro et al., 2013; Berzunza-Cruz
et al., 2015). Despite the attempts else where (Millan et al., 2014;
Rotureau et al., 2006; Rajendran et al., 1985; Mutinga, 1975; Morsy
et al., 1987), the extent of Leishmania natural infection in the Old
World bats remains uncertain, and cases of Chiropteran Leishmania
infections have not been documented in Ethiopia until now. In view
of these facts we carried out a Leishmania DNA survey in Ethiopian
bats.
2. Materials and methods
2.1. Sample collection
Bats were collected as a part of an extensive ecological and faunistic study in Ethiopia. Permission for trapping was obtained from
the Ethiopian Wildlife Conservation Authority (EWCA), government of Ethiopia. Here, we reported results for the 163 specimens
collected in leishmaniasis endemic (44 bats) and non-endemic (119
bats) areas of Ethiopia (Fig. 1). Bats were captured at presumed
flyways using a standard mist-net between 18:00 and 22:00 h.
Bats were removed from the net, anesthetized by intra peritoneal
injection of ketamine and xylazine. All the necessary external
morphological characters including size, color of hair and naked
parts, length of forearm, shape of snout, shape of ear and type of
membrane concerning the form of tail were recorded and the identification of each particular bat was confirmed based on the keys by
Happold and Happold (2013). Then bats were sacrificed and their
spleens were removed and kept in ethanol for the subsequent DNA
extraction.
167
2.2. DNA extraction, parasite detection and determination by PCR
All the techniques, materials and procedures: DNA isolation,
primers, real time polymerase chain reaction (RT-PCR) procedure,
target genes (kinetoplast DNA (kDNA) and 18S rRNA internal transcribed spacer one (ITS1)) and post PCR evaluation and parasite
determination, were performed as described in our previous work
on rodents (Kassahun et al., 2015). Briefly, for the purpose of Leishmania detection and identification, we tested extracted DNA using
RT-PCR targeting kDNA of Leishmania and positivity was confirmed
by direct sequencing of amplicons. Real time PCR targeting kDNA
gene is generally considered to be highly sensitive (Selvapandiyan
et al., 2008; Selvapandiyan et al., 2008) but sequence does not identify the Leishmania species (Nicolas et al., 2002; Nasereddin et al.,
2008). Therefore, all the kDNA positive specimens were re-analyzed
by RT-PCR of the ITS1 locus and positive samples underwent
sequencing of amplicons (Schoenian et al., 2003; Schoenian et al.,
2003).
3. Results and discussion
A total of 163 bats, belonging to 25 species of 18 genera (Table 1),
were collected. The dominant species in our collection were Pipistrellus hesperidus (18%), Miniopterus africanus (11%) and Scotoecus
hirundo (11%).
Amongst the 163 samples, Leishmania–kDNA positivity was confirmed by sequencing of a parasite DNA from eight bats belonging
to six species. Out of the eight Leishmania kDNA PCR positives,
the ITS1-PCR and subsequent sequencing revealed infection of L.
tropica in one specimen of Cardioderma cor and L. major in one specimen of Nycteris hispida (Table 1). We were unable to amplify ITS-1
sequences for the six additional Leishmania kDNA positive samples.
There was a similar scenario in our previous work (Kassahun et al.,
2015). PCR targeting kDNA fragment is considered to be highly sensitive due to the high number of target copies in each parasite cell.
Even though ITS-1 based PCR determines the species of the Leishmania parasite, the level of sensitivity is lower than that of kDNA PCR
(Abbasi et al., 2013) which does not provide sufficient information
for species determination.
Leishmaniasis due to L. tropica and L. major generally cause
dermal lesions in humans; however none of the bats had visible
dermal signs resembling cutaneous leishmaniasis. It is well know
that Leishmania species dermotropic for humans could migrate to
visceral organs of other animal hosts (Laskay et al., 1995). Moreover,
early dissemination of Leishmania parasites to the spleen has been
reported in asymptomatic animals (Schilling and Glaichenhaus,
2001). Such scenarios may explain our finding of parasite DNA
in the spleens of infected bats thus validating our experimental
approach for an epidemiological study.
Our finding represents a confirmed first report of natural
Leishmania infection of bats in the Old World. Previous studies conducted in the Old World (e.g. Spain (Millan et al., 2014), France
(Rotureau et al., 2006), India (Rajendran et al., 1985) and Kenya
(Mutinga, 1975)) did not yield any positive specimens. Moreover,
the attempts in Egypt (Morsy et al., 1987) were using old methods and the detection procedure was speculative with specificity
and parasite species characterization. However, bats in the New
World were repeatedly investigated and found infected with Leishmania species pathogenic to humans. In our study, the prevalence
reached 5% (8 out of 163) corresponds with the infection rates of
bats recorded in Sao Paulo, Brazil (4%) (Savani et al., 2010); while
higher prevalence has been detected in Venezuela (9%) (Lima et al.,
2008); Mexico (9.8%) (Berzunza-Cruz et al., 2015) and Mato Grosso
do Sul, Brazil (40%) (Shapiro et al., 2013).
168
A. Kassahun et al. / Acta Tropica 150 (2015) 166–170
Fig. 1. Map of leishmaniasis distribution in Ethiopia (modified and adapted from Leta et al., 2014; Seid et al., 2014 and unpublished hospital records) and trapping localities
with respective Leishmania DNA detection results.
Table 1
Bats collected in different trapping localitiesa in Ethiopia and examined for Leishmania DNA by RT-PCR. The number of Leishmania kDNA positive bats appears in square
brackets.
Bat species
Cardioderma cor
Glauconycteris variegata
Laephotis wintoni
Micropteropus pusillus
Miniopterus arenarius
Miniopterus africanus
Mops condylurus
Myotis scotti
Myotis tricolor
Neoromicia somalica
Neoromicia guineensis
Neoromicia nana
Nycteris hispida
Nycticeinops schlieffenii
Otomops martiensseni
Pipistrellus hesperidus
Pipistrellus rusticus
Rhinolophus fumigatus
Scotoecus hirundo
Scotophilus colias
Stenonycteris lanosus
Tadarida sp.
Triaenops afer
Total
BCH
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1
–
–
1
ABR
–
1
–
–
–
–
–
–
–
2[2]
–
–
–
3
–
–
–
–
3
–
–
–
–
9
MSH
–
–
–
–
2[1]
–
–
–
–
–
–
–
–
–
–
2
–
–
–
–
–
–
–
4
BDL
–
3[1]
–
2
–
–
–
2
–
–
3
–
–
–
–
–
–
–
1
1
–
–
–
12
DDS
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
8
–
8
KNS
–
–
–
–
–
–
–
–
–
2
–
–
–
–
–
–
–
–
14
–
–
–
–
16
TPI
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1
–
–
–
–
–
–
1
GOB
–
–
–
–
–
–
–
–
–
–
–
–
–
–
4
–
–
–
–
–
–
4
AMR
b
1 [1]
–
–
–
–
–
–
–
–
–
–
–
1 [1]c
–
–
–
–
–
–
–
–
–
–
2
ALM
WLT
MTU
SFO
SOR
MNG
˙ (%)
–
–
–
–
–
–
–
–
–
1
–
–
–
–
–
–
–
–
–
–
–
–
–
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1
–
–
–
–
–
1
–
4
–
2
–
–
9
–
–
–
–
–
–
–
–
9
–
–
–
11[2]
–
–
–
35
–
–
–
–
–
18
–
–
11
–
–
–
–
–
3
–
–
5
–
–
–
1
11
49
–
–
–
2
–
–
–
–
–
–
–
–
–
–
–
2
–
–
–
–
–
–
–
4
–
–
2
–
–
–
–
–
–
1
–
1
–
–
–
13
–
–
–
–
–
–
17
1 (0.6)
8 (4.9)
2 (1.2)
6 (3.7)
2 (1.2)
18 (11.0)
9 (5.5)
2 (1.2)
11 (6.7)
6 (3.7)
3 (1.8)
1 (0.6)
1 (0.6)
3 (1.8)
3 (1.8)
30 (18.4)
1 (0.6)
6 (3.7)
18 (11.0)
12 (7.4)
1 (0.6)
9 (5.5)
11(6.7)
163
a
Abbreviation of localities: BCH-Bechu, ABR-Aba-Roba, MSH-Masha, BDL-Bedele, DDS-Dedesa, KNS-Konso, TPI-Tepi, GOB-Goba, AMR-Awash-Methara, ALM-Alemata,
WLT-Welenchiti, MTU-Metu, SFO-Sof Omar caves, SOR-Sorr, MNG-Menagesha.
b
L. tropica positive bats.
c
L. major positive bats.
Four of the positive bats were captured in the Aba-Roba and
Awash-Methara leishmaniasis endemic foci while the other four
specimens originated from non-endemic localities of Metu, Bedele
and Masha (Fig. 1). The Awash-Methara foci are known for L. tropica infections in humans (Hailu et al., 2006a), phlebotomine sand
flies (Gebre-Michael et al., 2004) and recently rodents (Kassahun
et al., 2015). Our results corroborate these findings as one specimen
of C. cor captured in this area was found infected with L. tropica.
Although L. tropica is regarded to be anthroponotic, infections in
dogs (Baneth et al., 2014), golden jackal and red foxes (Talmi-Frank
et al., 2010) and rodents (Svobodova et al., 2003; Talmi-Frank et al.,
2010) have been well documented generally in zoonotic foci (Sang
A. Kassahun et al. / Acta Tropica 150 (2015) 166–170
et al., 1994). The finding of this parasite both in bats and in our previous study of rodents (Kassahun et al., 2015) points to the possibility
of zoonotic transmission in the particular area.
The specimen of N. hispida infected with L. major was trapped in
the same area, Awash-Methara. The findings of L. major in Ethiopia
are rare but natural infections in humans (Abbasi et al., 2013) and
sand flies (Gebre-Michael et al., 1993) were recorded in North and
South-west Ethiopia, respectively. No previous L. major infection
was reported in Awash-Methara region; however our unpublished
preliminary entomological survey in this area revealed the presence of Phlebotomus papatasi and Phlebotomus duboscqi, both being
considered as a potential vectors of L. major (Dostalova and Volf,
2012).
The finding of four Leishmania–kDNA positive bats in the
non-endemic localities could be explained by the fact that the geographical distribution of the parasite in Ethiopia is much wider than
anticipated. Moreover, bats have a potential to migrate from place
to place and we could hardly rule out the possibility that bats from
Leishmania endemic areas could move to non-endemic areas.
No L. donovani complex DNA was detected in our bats sample. It is obvious that L. donovani is the sole agent of human VL
in Ethiopia with wide geographical areas (Hailu et al., 2006a). The
recent finding of DNA in rodents (Kassahun et al., 2015) and domestic animals (Rohousova et al., 2015) could also determine its host
range. However, the absence of this species in bats doesn’t reflect
being refractory or the parasite’s specificity.
Generally, to determine the role of a given host in a reservoir
system it should fulfill some criteria among others: overlap of geographical distribution of vectors and hosts; forming large biomass,
being gregarious and long lived in addition to being found naturally
infected and subsequently being infective for transmitting vectors
(Ashford, 1996). Some of these conditions work with bats and their
ability to fly long distances and colonize places could make them
suitable bridge hosts for leishmaniasis. Moreover, most colonies of
bats live and rest in caves and cracks that are assumed to provide
ambient temperatures and relative humidity suitable for sand fly
breeding and diurnal resting (Feliciangeli, 2004). Laboratory feeding experiments on Lutzomyia longipalpis, most widely distributed
vector of New World VL, was capable of feeding from different families of bats that suggested the importance of bats as a possible
natural blood source of sand flies (Lampo et al., 2000). In addition
to this, bats are well known hosts of Trypanosoma transmitted by
sandflies (McConnell and Correa, 1964; Williams, 1976; Lord and
Brooks, 2014), which is closely related to the genus Leishmania.
In conclusion, bats could have adequate features to be naturally infected by Leishmania and could subsequently to play a role
in its epidemiological cycle. The present study revealed natural
Leishmania infections of Old World bats, in areas both endemic
and non-endemic for human leishmaniasis. The wide geographical distribution of Leishmania parasite in the country could imply
the existence of different modes of transmission and our finding
might indicate the importance of bats in the disease cycle. However,
to play a role in Leishmania cycles it is required to investigate the
host’s pathogenic features and being infectious to vectors; which
were not covered in this paper. Thus, further studies on persistence of the Leishmania parasite in bats and its interaction with
sand fly vectors are recommended for the better understanding of
their epidemiological involvement.
Acknowledgements
This project was funded by grants from the Bill and Melinda
Gates Foundation Global Health Program (OPPGH5336), Grant
Agency of the Charles University in Prague (GAUK 9108/2013) and
the EU grant 2011-261504 EDENext (the paper is catalogued as
169
EDENext 427). The funding agencies had no role in study design,
data collection and analysis, decision to publish, or preparation of
manuscript.
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