ticks (acari: argasidae, ixodidae) parasitizing bats in the central balkans
TRANSCRIPT
Ticks (Acari: Argasidae, Ixodidae) parasitizing batsin the central Balkans
J. Burazerovic • S. Cakic • D. Mihaljica •
R. Sukara • D. Cirovic • S. Tomanovic
Received: 2 December 2014 / Accepted: 17 February 2015� Springer International Publishing Switzerland 2015
Abstract Ticks parasitizing bats have been largely understudied, especially in the central
part of the Balkan Peninsula, where the last data from the field research date from almost
25 years ago. Bats are hosts to a large number of ectoparasites, including ticks, which can
act as vectors of zoonotic agents. For this reason, it is important to identify the distribution
of ticks and their relationship to different hosts, including wild animals, bats in particular.
The present research was conducted at 16 localities throughout Serbia, Montenegro, Bosnia
and Herzegovina, and Former Yugoslav Republic of Macedonia (FYROM). We examined
475 individuals of bats belonging to 13 species. A total of three tick species were iden-
tified, I. simplex being the most numerous and widespread, followed by I. vespertilionis
and A. vespertilionis. To the best of our knowledge, the presented data include the first
records of I. simplex in Serbia and Montenegro, I. vespertilionis for Montenegro and
A. vespertilionis in FYROM. Also, we identify a new possible host/parasite association
between I. simplex and Rhinolophus euryale.
Keywords Bats � Ticks � Balkan Peninsula � Ixodes sp. � Argas sp.
Introduction
Bats, with over 1116 species described (Simmons 2005) represent around a quarter of all
mammalian species on Earth. They are increasingly recognized as reservoirs of emerging,
mostly zoonotic, viral (Calisher et al. 2006) and bacterial (Muhldorfer 2013) pathogens.
Bats are hosts to a large number of ectoparasites, including ticks, which can act as vectors
J. Burazerovic � D. CirovicChair of Animal Ecology and Zoogeography, Faculty of Biology, University of Belgrade, Belgrade,Serbia
S. Cakic � D. Mihaljica � R. Sukara � S. Tomanovic (&)Laboratory for Medical Entomology, Department of Parasitology, Institute for Medical Research,University of Belgrade, Belgrade, Serbiae-mail: [email protected]
123
Exp Appl AcarolDOI 10.1007/s10493-015-9891-6
of zoonotic agents. Due to their specific migrating behaviour (Hutterer et al. 2005; Flaquer
et al. 2009), bats can cross barriers and move long distances in a short time, transmitting
ticks and tick-borne pathogens.
Three, possibly four, hard tick species have adapted to live exclusively on bats. All of
them belong to the genus Ixodes. Ixodes simplex Neumann, 1906 is found mainly on
Miniopterus schreibersii, I. kopsteini Oudemans, 1926 lives on mastiff bats (Tadarida sp.),
and I. vespertilionis Koch 1844 parasitizes different bat species. All three species are found
in the Old World and Australia, while in Europe I. simplex and I. vespertilionis are
recorded (Kolonin 2007), together with the recently described species I. ariadnae Hornok
et al. 2014, collected from caves and bats in Hungary (Hornok et al. 2014). Accidental
findings of non-specific I. ricinus and I. trianguliceps have been recorded on bats in Poland
and Slovakia (Siuda et al. 2009; Sevcik et al. 2010). In the family Argasidae, Argas
vespertilionis Latreille, 1796 is a common tick recorded as a parasite of Old World bats
(Hoogstraal 1985; Siuda et al. 2009; Manzano-Roman et al. 2012; Petney et al. 2012).
Data on bat ticks from countries geographically situated in the central part of the Balkan
Peninsula—Bosnia and Herzegovina, FYROM, Montenegro and Serbia—are very scarce
and fragmented, and only a few published records are available in the literature. Most of
the country-specific data actually derive from the time when these countries were united in
the former Yugoslavian federation (Oswald 1940; Tovornik 1990). Furthermore, borders in
this part of Europe have changed frequently in the past, which makes identification of exact
locations even harder, especially if data derive before the time of the former Yugoslavian
federation (Karaman 1937; Vrenozi and Dunlop 2013).
The aim of this study is to fill in the gaps in knowledge about distribution of bat ticks in
the Central Balkans as well as to provide more information about host-parasite interactions
between cave-dwelling bats and ticks. Here we present new records of bat tick species, as
well as a new parasite/host association, from the central Balkan Peninsula. Along with
morphological identification of tick species, the research included applied molecular
analysis of the cytochrome oxidase subunit I (COI) gene as a complementary method to the
rather vague and incomplete available taxonomic keys and descriptions of bat tick species.
Materials and methods
Collection of data
The field work was conducted during the period April–October 2013, extensive study
being suspended in late May through June until late July during the period of advanced
pregnancy, delivery, and the first phase of nursing the young bats. Bats were caught using a
mist net placed at cave entrances, while bats in their roosts (attics, cellars, caves) were
taken by hand or with hand net, and released at the same site where caught. All bats caught
were identified to the species level (Dietz et al. 2009). The whole body of the host was
carefully examined, and any ticks found were removed with tweezers and placed in tubes
with 70 % ethanol, and labelled appropriately.
Ticks were separated by developmental stage and gender (adults) and identified to the
species level using morphological keys (Pomerancev 1950; Arthur 1956). The preserved
parasites are deposited in collections of the Laboratory of Medical Entomology, Institute
for Medical Research, University of Belgrade.
Exp Appl Acarol
123
DNA isolation, amplification, sequencing, and sequence analysis
In order to confirm morphological identification of Ixodes species which was based on the
rather vague and incomplete available taxonomic keys, randomly selected tick samples
were chosen for further molecular analysis. DNA was extracted from whole ticks or legs,
using Kapa Express Extract Kit (Kapa Biosystems) according to manufacturer’s instruc-
tions. DNA extracts were stored at -80 �C until PCR amplification.
For amplification of cytochrome oxidase subunit I gene (COI) universal primers LCO1490
(forward: 50 GGTCAACAAATCATAAAGATA TTGG 30) and HCO2198 (reverse: 50
TAAACTTCAGGGTGACCAAAAAATCA 30) were used (Folmer et al. 1994). PCR reac-
tion was performed with 2 ll of extracted DNA as template. Each 50 ll reaction consisted of
17 ll H2O, 25 ll of Kapa 2G Robust Hot Start Ready Mix (Kapa Biosystems) and 3 ll of
each of the two primers. An initial denaturation at 94 �C for 3 min was followed by ten cycles
(denaturation at 95 �C of 15 s., annealing at 49 �C for 15 s and extended at 72 �C for 15 s.),
additional 35 cycles (denaturation at 95 �C of 15 s., annealing at 53 �C for 15 s and extended
at 72 �C for 15 s.) and final extension at 72 �C for 10 min. To confirm the efficiency of
amplification, products were analysed by 2 % agarose gel electrophoresis.
Products proven to be positive after agarose gel electrophoresis underwent sequencing.
DNA sequencing, including primer walking was performed by Macrogen Inc. Amsterdam
Netherlands. The representative sequences were deposited in the GenBank database under
accession numbers (I. simplex KJ997948, KM062041; I. vespertilionis KM062042,
KM062043)
Fig. 1 Map with visited localities: open circle locality with no recording of any tick species, closed circlelocality with Ixodes vespertilionis present, closed triangle locality with I. simplex present, closed squarelocality with I. vespertilionis and I. simplex present, closed star locality with Argas vespertilionis present
Exp Appl Acarol
123
The standard nucleotide blast tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to
align obtained sequences with mtDNA sequences available in GenBank. Additional align-
ment was performed using Clustal W Multiple Alignment (Thompson et al. 1994). For
phylogenetic analysis, additional mtDNA COI sequences of I. vespertilionis and I. simplex
originating from the geographically closest locations (Hungary) were included, together with
taxonomically related Ixodes species (I. ariadnae, I. lividus, I. redikorzevi and I. ricinus)
available in GenBank. Preliminary phylogenetic analyses were performed with MEGA 5.2
(Tamura et al. 2011) using both the Neighbor-joining and maximum likelihood tree recon-
struction methods. Genetic distances between sequences were calculated by Kimura’s two-
parameter method (K2P) of base substitution. In phylogenetic analyses, the Rhipicephalus
sanguineus mtDNA COI sequence was used as an out group (GenBank KF437543).
Results
Data were gathered at 16 localities in four countries of the central part of the Balkan
Peninsula: Serbia, Montenegro, Bosnia and Herzegovina, and FYROM (Fig. 1). A total of
475 individuals belonging to 13 bat species were collected and examined: Rhinolophus
Fig. 2 Relationship between bat and tick species with corresponding number of samples recorded in thisresearch. The size of the circles correlates with the number of bat/tick samples. The thickness of the arrowsindicate number of ticks parasitizing on the specific bat species
Exp Appl Acarol
123
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Exp Appl Acarol
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euryale, R. ferrumequinum, R. hipposideros, M. blythii, Myotis capaccinii, M. daubentonii,
M. emarginatus, M. myotis, M. mystacinus, Nyctalus noctula, Pipistrellus pipistrellus,
Plecotus austriacus, Miniopterus schreibersii. The highest diversity of bats was found in
western Serbia (Petnicka pecina cave—eight species, Deguricka pecina cave—five species
and Salitrena pecina cave—five species) and Montenegro (Zacirska pecina cave—five
species and Jama Sutonjica cave—five species). Ticks were found on five species of bats:
R. euryale, R. ferrumequinum, M. schreibersii, M. mystacinus and P. pipistrellus. Out of 16
visited localities, bats with ticks were found at nine. Ticks were found at six out of seven
localities in Serbia, two out of three localities in Montenegro, and one out of three lo-
calities in FYROM. No ticks were recorded on bats caught in Bosnia and Herzegovina.
Overall, 94 individuals (19.79 %) of the total number of examined bats were parasitized by
one or more tick individuals. The usual predilection sites of ticks on bats were: around the
mouth, around the eyes, on and around the ears, and on the dorsal and ventral side of the
body close to the patagium. A total of 165 ticks were collected: 158 specimens of Ixodes
simplex (18 females, 92 nymphs, and 48 larvae); six specimens of Ixodes vespertilionis
(one female, two nymphs and three larvae) and one larva of Argas vespertilionis. Ixodes
simplex was found on two bat species—M. schreibersii (156/158 ticks) and R. euryale (2/
158); I. vespertilionis was recorded on four bat species—R. euryale (3/6 ticks), R. fer-
rumequinum (1/6), M. schreibersii (1/6) and M. mystacinus (1/6); while a single specimen
of A. vespertilionis was found on P. pipistrellus (Fig. 2).
The prevalence of animals infested with I. simplex was the highest among bats of the
species M. schreibersii (87/281–30.96 %), while we found only one larva on one R.
euryale (1/39–2.56 %). The bat species most frequently infested with I. vespertilionis was
Fig. 3 Phylogenetic relationship of Ixodes simplex and I. vespertilionis COI gene sequences obtained in thepresent study (bold) in comparison with representative Ixodes spp. and Rhipicephalus sanguineus sequencesavailable in the GenBank
Exp Appl Acarol
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M. mystacinus (1/6–16.66 %), followed by R. euryale (3/39–7.69 %), R. ferrumequinum
(1/21–4.76 %) and M. schreibersii (1/281–0.36 %) (Table 1).
Molecular analysis was successful for three specimens of I. simplex and two specimens
of I. vespertilionis. Sequencing yielded four different genotypes (two genotypes of I.
simplex and two genotypes of I. vespertilionis). Molecular and phylogenetic analysis
confirmed the standard morphological identification and taxonomic status of the analyzed
ticks (Fig. 3).
Discussion and conclusion
The findings presented in this paper reveal new aspects of the distribution and host range of
ticks parasitizing bats from the central part of the Balkan Peninsula. Our data include the
first records of I. simplex in Serbia and Montenegro, I. vespertilionis in Montenegro, and A.
vespertilionis in FYROM.
The most widespread tick species collected during this study was I. simplex (six out of
16 localities), I. vespertilionis was collected at five out of 16 localities, while A. vesper-
tilionis was recorded only at one location in the western part of FYROM, in a habitat that
was not a cave – it was found on a common pipistrelle bat caught in a mist net in an old
orchard. All other ticks were collected from bats caught in caves. Previous data on bats of
the families Vespertilionidae and Rhinolophidae in Europe indicated that I. vespertilionis is
the dominant species (Arthur 1956, 1963; Walter and Kock 1985), while information about
I. simplex remained mostly fragmented and scarce. In our research I. simplex is both the
most widespread and the most numerous species.
Starting from the beginning of the twentieth century, in the territory of former Yu-
goslavia, I. vespertilionis was reported in all republics except Montenegro (Neumann 1916;
Oswald 1940; Arthur 1956; Tovornik 1990). The presence of the species was documented
in the following surrounding countries as well: Hungary (Neumann 1916; Arthur 1956;
Siuda et al. 2009; Hornok et al. 2012), Bulgaria (Arthur 1956; Beron et al. 2011), and
Romania (Mihalca et al. 2012). Our records of this species for Montenegro confirmed
assumptions of its presence there based on its previously observed wide distribution in the
region.
As hosts of I. vespertilionis, we recorded M. schreibersii (Salitrena pecina cave),
R. euryale (Petnicka and Zacirska pecina caves, Jama Sutonjica cave), Rhinolophus fer-
rumequinum (Petnicka pecina cave) and M. mystacinus (Lazareva pecina cave) - all species
previously identified as hosts. In Europe most records of I. vespertilionis are from bats
belonging to the genus Rhinolophus (R. ferrumequinum, R. hipposideros, R. euryale), but it
is also recorded from other species (M. myotis, M. blythii, M. mystacinus, M. alcathoe, M.
daubentonii, M. schreibersii, Plecotus auritus, Nyctalus noctula and Pipistrellus pygmeus).
Thus it appears to be less host-specific than I. simplex (Arthur 1956; Danko et al. 2010;
Sevcik et al. 2010; Mihalca et al. 2012; Petney et al. 2012). Previous data for Serbia
(Tovornik 1990) reported two locations in the eastern part of the country, where specimens
were collected only from cave walls. During our research the ticks were collected for the
first time directly from bats, confirming suspected host species.
Concerning I. simplex, up to our research there was no reliable published information
indicating its presence in this part of the Balkan Peninsula. Confusion is caused by the
inconstancy of borders between the former Yugoslav republics. Karaman (1937) refers to
findings of I. simplex in ‘‘southern Serbia’’, but this locality most likely belongs to the
recent FYROM.
Exp Appl Acarol
123
Ixodes simplex is known to inhabit caves and feeds exclusively on bats, especially M.
schreibersii. Beaucornu (1967) and Haitlinger and Rupert (1985) cited M. schreibersii as
the main host of this tick species. However, several authors reported the given tick from
other host taxa as well, e.g., R. ferrumequinum (Nuttall et al. 1908), M. myotis (Walter and
Kock 1985) and Rhinolophus affinis (Kolonin 2003). According to Sevcik et al. (2010), I.
simplex was found on M. myotis in Poland, which is an area outside the range of its main
host, M. schreibersii (Haitlinger and Rupert 1985). They also report I. simplex on Rhi-
nolophus hipposideros, but assume it to be an occasional host species. The specimens
collected in the present study were mainly parasitizing M. schreibersii in Serbia. At one
locality in Montenegro (Jama Sutonjica cave), we collected a specimen of I. simplex from
R. euryale, indicating a possible new host/parasite association. To the best of our
knowledge, the presence of I. simplex on this particular bat species has not been recorded
to date and could be explained by a transfer from its main bat host, M. schreibersii, whose
presence was recorded in the same roost where the affected individual of R. euryale was
found.
The absence of ticks on bats collected at three localities in Bosnia and Herzegovina can
be attributed to the small number of bats caught for tick collection and the fact that they
belonged to species not identified as hosts of any ticks in this study (R. hipposideros and M.
blythii).
The majority of collected samples were nymphal and larval ticks, so we performed
molecular analysis of barcoding mtCOI sequences from representative I. simplex and I.
vespertilionis specimens in order to confirm morphological identification based on keys of
Pomerancev (1950) and Arthur (1956) lacking data on subadult stages or presenting only
free-hand drawings. The sequencing results confirmed morphological identification and
taxonomic status, but since we noticed a certain level of heterogeneity (five sequences
yielded four different genotypes) in the analyzed sequences, further phylogeographic study
is planned.
The present paper also represents the first reliable finding of A. vespertilionis for
FYROM, as in the literature only vague information exists in a work giving the Regional
disease vector ecology profile for Central Europe (www.afpmb.org/sites/default/files/pubs/
dveps/CentralEurope.pdf) with no reference to any paper, author, locality, or date of
finding this tick species. Argas vespertilionis is a widely distributed tick species mainly
specialized for feeding on bats as hosts and occurs throughout Europe, Asia, and Africa,
ranging from the British Isles in the west to Japan, Korea, and India in Asia (Siuda et al.
2009; Petney et al. 2012). Hosts are predominantly bats (Siuda et al. 2009), the genus
Pipistrellus having been identified as the preferred one. It is found on 14 of 25 Poland’s bat
species and on five of 28 Slovak bat species (Siuda et al. 2009), including both cave-
dwelling and forest bats. According to Siuda et al. (2009), A. vespertilionis occurs in
breeding and transient colonies, attics, burrows, tree holes, and caves. Due to this wide
scope of habitats, it can be expected to occur on a large number of bat species. It also
parasitizes bats in synanthropic and semi-synanthropic habitats, where it hides in the cracks
of walls of buildings and caves, as well as under piles of guano (Nowak-Chmura and Siuda
2012).
Argas vespertilionis is known to attack humans and domestic animals and has been
reported to be ‘‘highly aggressive’’ (Hoogstraal 1985; Estrada-Pena and Jongejan 1999).
Another important feature of this tick species is that it has been associated with several
pathogens of human and animal importance: Issyk Kul virus and Sokuluk virus (Hoogstraal
1985; Gavrilovskaya 2001; de la Fuente et al. 2008), Borrelia burgdoferi sensu lato
(Hubbard et al. 1998),Coxiella burnetii and Wolbachia sp. (Nowak-Chmura and Siuda
Exp Appl Acarol
123
2012). Evans et al. (2009) reported a fatal case of relapsing fever in a pipistrelle bat
(Pipistrellus sp.) in the United Kingdom caused by a new and unknown Borrelia species,
assumed to be transferred to the bat by a tick of the species A. vespertilionis found attached
to the bat. Socolovschi et al. (2012) analyzed five specimens of this tick species and found
a new genotype of Rickettsia sp., new species of the Ehrlichia canis group and Borrelia sp.
CPB1, a relapsing fever agent of the Borrelia group that caused the fatal borreliosis in a bat
in the United Kingdom previously reported by Evans et al. (2009).
To judge from the scarce data available, the medical importance of the recorded Ixodes
species appears to be minor. However, Hornok et al. (2012) isolated for the first time
Bartonella sp. from a female I. vespertilionis, implying an association of bartonellae with
Rhinolophus sp. and M. myotis. Although known to be specialized for feeding on bats, the
recently reported first case of human infestation by I. vespertilionis indicates the potential
medical importance of this tick species (Piksa et al. 2013). As for I. simplex, no data
indicating any medical significance have been found so far.
From the viewpoints of public health and conservation medicine, it is necessary to
emphasize the importance of wildlife species known to be tick hosts and potential tick-
borne pathogen reservoirs as sentinel species ideal for monitoring the presence of tick-
borne diseases in specific ecosystems. The role of bats with their specific biology and
behaviour as potential reservoirs of vector-borne pathogens, especially viruses, is coming
into focus. Further systematic studies are required to discern the enzootic cycles of tick-
borne pathogens involving bats as host and reservoir species. An awareness on the part of
professionals and recreationists visiting specific bat habitats, as well as residents, veteri-
narians, and health professionals in areas where synanthropic bat species occur—would be
of great importance for efficient monitoring of these zoonotic diseases.
Acknowledgments This work was supported by a grant from the Ministry of Education, Science andTechnological Development of the Republic of Serbia (Project No. 173006). The work of ST was doneunder the frame of EurNegVec COST Action TD1303. The authors wish to express their gratitude to allcollaborators in the field who helped in identification of bat roosts and field work for this study: MilosPavicevic, Marina Ðurovic and Veso Mijuskovic from Montenegro, Dejan Radosevic, Mladen Samardzicand Jelena Golijanin from Bosnia and Herzegovina, Darko Dragulovic, Petar Petrovic, Nina Boskovic andstudents of the Faculty of Biology at the University of Belgrade from Serbia.
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