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A. Diakou, E. Kapantaidakis and D. Youlatos
Endoparasites of the European ground squirrel (Spermophilus citellus)
(Rodentia: Sciuridae) in central Macedonia, Greece
825025
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Journal of Natural History, 2013
Vol. 00, No. 00, 1–12, http://dx.doi.org/10.1080/00222933.2013.825025
RESEARCH ARTICLE
Endoparasites of the European ground squirrel (Spermophilus citellus)
(Rodentia: Sciuridae) in central Macedonia, Greece
AQ1
Q1
A. Diakoua* , E. Kapantaidakisa and D. Youlatosb
a
Laboratory of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, Aristotle
Ar
University of Thessaloniki, Thessaloniki 54124, Greece; b Department of Zoology,
gy, Schoo
School of
Biology, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
5
(Received 8 November 2012; ﬁnal version received 17 April 2013)
The European ground squirrel (Spermophilus citellus) is a small
smal rodent categocat
rized as vulnerable (IUCN). To investigate the parasitic
in
sitic fauna of this
t is species
spe
Greece, faecal samples from 125 animals belonging to six different populations
popu
were
examined by standard parasitological methods. Parasites
Parasit s were found iin 118 of the
animals (94.4%). Oocysts of the coccidia Eimeria
were found in
meria callospermophili
callospermop
92 animals (73.6%), Eimeria citelli in 76 (60.8%),
8% Eimeria
ia cynomysis
c omy in 41 (32.8%),
Eimeria spp. (17.6%), Cryptosporidium spp. in
of Entamoeba spp.
n 29 (23.2%), cysts
c
in 32 animals (25.6%) and eggs of the trematode
atode Brachylaima
chyl
spp. in seven animals (5.6%). This is the ﬁrst reportt of Entamoeba
Cryptosporidium spp. and
eba spp., C
Brachylaima spp. in S. citellus. The
possible impact of these ﬁndings on the health
he possib
status of S. citellus and the possible
to domestic animals or public
ossible signiﬁcance
sign
health is discussed.
10
15
20
Keywords: Spermophilus
us citellus; parasites;
pa asites protozoa; trematoda; Greece
Introduction
The European ground
round squirrel
squirr or European souslik (Spermophilus citellus) is a small
mammal belonging
Rodentia and the family Sciuridae. It is a diurnal
ing to the order
or
iving in colonies of individual burrows, usually in short grass open habitats
animal, living
tufek and V
ralik 2005). The European ground squirrel is endemic to central
(Kryštufek
Vohralik
outheastern Euro
and southeastern
Europe (Figure 1) (Kryštufek 1999; Kryštufek and Vohralik 2005;
Ozkurt eett al. 22005), reaching its southernmost range in northern Greece, and has been
categoriz
categorized as vvulnerable according to the International Union for the Conservation
of Natu
Nature (Coroiu et al. 2008).
Al
Although the ecology, morphology and physiology of the European ground squirrel have been adequately studied (e.g. Fraguedakis-Tsolis 1977; Kryštufek 1993, 1995;
Huber et al. 1999; Millesi et al. 1999; Hut et al. 2002; Katona et al. 2002; Ozkurt
et al. 2002; Hoffmann et al. 2003; Everts et al. 2004; Vaczi et al. 2006; Youlatos et al.
2007), investigations concerning the parasites of this species are scant (Stefanov et al.
2001; Golemansky and Koshev 2007, 2009). However, the role of parasitism in wildlife
could be of great importance, especially in peripheral populations that are seriously
*Corresponding author. Email: diakou@vet.auth.gr
© 2013 Taylor & Francis
25
30
35
2 A. Diakou et al.
Figure 1. The range of Spermophilus
us citellus
cit
in Europe,
Eu
according to IUCN.
declining. This is the case for the species
s
in northern Greece, where populations are
isolated by intense
nse urbanization
urbanization and habitat degradation, allowing little or no contact,
viously ad
ent unit
between previously
adjacent
units. Such units are subject to inbreeding and vulnerine and extinction.
extincti
able to decline
Therefore, their welfare and health are of outmost
ance for
for their
thei protection and conservation.
importance
In this con
xt, th
context,
the aim of the present study was to investigate the endoparasitic
na of different
differ
fauna
populations of S. citellus, for the ﬁrst time in Greece, to enrich our
edge of
o the species’ health status and of potential threats to its conservation, as
knowledge
cons
well as to consider
any potential consequences of its parasites for domestic animals or
h
public health.
Material and methods
Study sites and animals
The current study was conﬁned to central Macedonia, Greece, where most of the
populations of the species currently occur. For technical reasons, we initially located
colonies of S. citellus in areas within the prefecture of Thessaloniki. The colonies
40
45
50
Journal of Natural History 3
Figure 2. The sampling sites
tes of Spermophilus
Spermoph
citellus in central Macedonia, Greece
(1: University Agriculture Farm,
Army Ve
Veterinary Hospital, 3: Axios – eastern coast, 4:
arm, 2: C Arm
Axios – western mound, 5: Anatoliko, 6: Aggelochori
salt pits).
Ag
were identiﬁed byy the presence
presenc of characteristic burrow entrances with signs of activd them (freshly excavated
exc
ity around
soil, food remains and droppings). In this way,
ntiﬁed and sampled six different populations from an equal number of areas
we identiﬁed
from central Macedon
Macedonia (Figure 2). These populations were selected mainly because
he distance
distance between them, as well as natural (e.g. deep rivers, forested areas) and/or
the
anthropo
enic ((e.g. highways, frequently used roads, extended urban areas) barriers
anthropogenic
assured their di
differentiation. The number of sampled burrows per population varied
betwe
between 5 and 37 (Table 1).
55
60
Faecal samples
Faecal samples were collected from the entrances of burrows. All the faecal pellets
of a single entrance were considered an individual sample and were collected in a
latex glove. As each burrow is used exclusively by one animal, each sample was considered to correspond to a single individual. For every burrow sampled, the exact
location was recorded, using a portable GPS (Garmin etrex, Garmin Inc., Olathe,
KS, USA).
65
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AQ2
4 A. Diakou et al.
Table 1. Spermophilus citellus sampling sites, number of burrows examined from each site and
parasites found in each site.
Sampling site
1. University Agriculture Farm
No. of burrows
sampled
Parasites
36
Eimeria callospermophili
E. citelli
E. cynomysis
Cryptosporidium spp.
2. C Army Veterinary Hospital
33
E. callospermophili
E. citelli
E. cynomysiss
Cryptosporidium spp.
3. Axios – eastern coast
19
E. callospermophili
lospe
E. citelli
cite i
E. cynomysis
ynom sis
Eimeria spp.
spp
Entamoeba spp.
sp
Cryptosporidium
spp.
Cryp
Brachylaima
spp.
B achyl
4. Axios – western mound
14
E. callospermophili
E.
E citelli
E. cynomysis
Eimeria spp
Entamoeba spp.
Cryptosporidium spp.
5. Anatoliko
188
E. callospermophili
E. citelli
E. cynomysis
Eimeria spp.
Entamoeba spp.
Cryptosporidium spp.
6. Aggelochori salt pits
5
E. callospermophili
Entamoeba spp.
AQ7
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Faecal
al examination
examin
All faecal
samples were examined using standard parasitological methods, i.e. zinc
al sam
ota
sulphate ﬂotation,
Telemann sedimentation and Ziehl–Neelsen-stained smears as
described before, with minor modiﬁcations (Henriksen and Pohlenz 1981; MAFF
1986; Thienpont et al. 1986). Initially the faecal material of each burrow was well
mixed and homogenized. For the zinc sulphate ﬂotation method, approximately 1 g
of the homogenized faecal material was diluted with tap water and passed through a
sieve (No. 150) in a centrifuge tube. The tube was centrifuged at 1500 rpm for 3 min,
then the supernatant ﬂuid was discharged down to approximately 1 cm above the sediment and zinc sulphate (ZnSO4 · 7H2 O) solution 33.2% (weight/volume) was added
to the sediment. After thorough dilution of the sediment, zinc sulphate solution was
70
AQ
Q
75 AQ3
Journal of Natural History 5
added to just over the top of the tube and a cover slip was placed on the top of the
Q
AQ4
tube. After centrifugation at 900 rpm for 1 min, the cover slip was carefully removed,
80 AQ
placed on a microscope slide and examined under the optical microscope at 100×
and 400× magniﬁcation. For the Teleman sedimentation method, approximately 1 g
of the homogenized faecal material was diluted in HCl 16%, passed through a sieve
(No. 150) in a centrifuge tube, 5 ml ether was added and the content of the tube was
AQ5
Q
homogenized by vigorous shaking. After centrifugation at 1500 rpm for 3 min all the
85 AQ
phases of the centrifuged material but the sediment were discharged. Drops of the
sediment were examined under the optical microscope at 100× and 400× magniﬁcation. For the Ziehl–Neelsen-stained smears, faecal smears were prepared
ed on glass
slides, ﬁxed by passing over the ﬂame of a Bunsen burner and covered with
th carbol–
fuchsin solution that was kept warm for 5 min. The smears were then
90
hen decolourized
decol
with alcohol–acid mixture, rinsed with tap water and ﬁnally covered for 1–2
–2 min with
Malachite green. After a last rinse with tap water, the smears
were allowed to dry
rs w
000 ) for the detection
det
and then examined under the microscope with oil lens (1000×)
of
Cryptosporidium spp. oocysts.
sed on the morphology
mo phol
The identiﬁcation of the parasites found was based
of their 95
Duszyn
reproductive elements in the faeces (Levine and Ivenss 1990; Duszynski
et al. 2001;
n was feasible
feasible to species level for some
Haralabidis and Diakou 2001). The identiﬁcation
ﬁcation to species
sp cies level for all parasites
parasites and to genus level for others. Identiﬁcation
ecropsy) or molecular
m
would require either adult parasites (after necropsy)
techniques, both
being beyond the scope of the present study.
100
Sporulation of coccidian oocysts
ontained unsporulated
unspor
The faecal samples that contained
coccidian oocysts were allowed to
morpho ogic characteristics, necessary for identiﬁcasporulate to fully develop their morphological
at purpose, the
th faecal material was left in a solution of 2.5%
tion of the species. For that
p
d
(weight/volume) aqueous potassium
dichromate
(K2 Cr2 O7 ) at room temperature and 105
ay for the completion
com l i of the sporulation (Boch and Supperer 1992).
checked every day
Resultss
rasites were found in 118 of the 125 sampled animals (94.4%). Coccidia of the genus
Parasites
Eimeria were
ere detected
d
in 116 of the animals (92.8%). More precisely, Eimeria callosperw found
foun in 92 animals (73.6%), Eimeria citelli in 76 animals (60.8%), Eimeria 110
mophili was
cynomy
cynomysis in 41 animals (32.8%) and two unidentiﬁed species of the genus Eimeria
in 22 aanimals (17.6%). In addition, two more protozoan genera were found: cysts of
moe spp. in 32 animals (25.6%) and oocysts of Cryptosporidium spp. in 29 aniEntamoeba
mals (23.2%). Finally, eggs of the trematode Brachylaima spp. were found in seven
animals (5.6%).
115
All the sampled colonies were infected with parasites. Mixed infections with
[more than one parasite species were common with various combinations of parasites. The most prevalent was the E. callospermophili and E. citelli co-infection,
found in 34 animals (27.2%). The greatest variety of parasites, i.e. E. callospermophili,
E. citelli, E. cynomysis, Cryptosporidium spp., Entamoeba spp. and Brachylaima spp., 120
was found in two animals (1.6%). The parasites found in the different sampling areas
6 A. Diakou et al.
are presented in Table 1. In 20% of the Eimeria-positive samples, the oocysts were in
abundance in the optic ﬁeld (> 20 oocysts/o.f. in ×100 magniﬁcation).
Discussion
The range of the European ground squirrel is divided by the Carpathian Mountains
into two main basins: the Pannonian and the Balkan. The northwestern area of its
distribution (Pannonian basin) covers parts of the Czech Republic, Austria, Slovakia,
Hungary, northern Serbia, Montenegro and western Romania, while the southeastern area of its range (Balkan basin) extends into parts of southern Serbia, Former
rm
Yugoslav Republic of Macedonia, northern Greece, Bulgaria, southern Romania,
ani
Turkish Thrace, Moldova and Ukraine (Panteleyev 1998; Kryštufek 1999).
Despite its relatively large range in central, eastern and southern Europe, popuast 10 years, with
h
lation numbers appear to have rapidly declined by > 30% in the last
ssful reintroductio
regionally extinct populations in Croatia and Germany and successful
reintroductions
wed tthat
hat most re
in Poland (Coroiu et al. 2008). However, Mat˘eju˚ et al. (2010) showed
reinen the current popu
troduction projects in central Europe had little success. Given
population
ations could be especially
status of the species across Europe and its range, Greek populations
important, as they are found at the periphery of the southern bord
border of the range of
ne in this area too
oo ((Youlatos 2009).
the species, and there is evidence of population decline
Therefore, the health of these peripheral populations is fundamen
fundamental for their survival,
as well as for future action plans on protection and conservation of the species across
tly rrelated to the hea
Europe. In this context, parasitism is directly
health of established populations and therefore, identifying and evaluating the parasitic load is important in
understanding the health robustness of a given po
population.
luatin parasitism in wild animals: (1) the
There are three main points when eva
evaluating
asites on the animal
nim its self, (2) the importance of these
effect and importance of the parasites
nd, (3) the pos
parasites to domestic animals and,
possible impact on public health (Choquette
mals, p
arasites are aalmost always present but apparently in
1956). In free-living animals,
parasites
ittle damag
e. Howev
most cases are doing little
damage.
However, under certain circumstances the host–
an be distur
parasite relationship can
disturbed and then a clinical condition develops. Such
ay b
circumstances may
be malnutrition
malnutrition, extreme weather conditions and co-existence of
ns. In ca
es of vulne
other pathogens.
cases
vulnerable or threatened animal populations, the role of
comes even mo
parasites becomes
more signiﬁcant.
esent study, the most prevalent parasites of S. citellus were the coccidian
In thee pr
present
zoa of the
the genus
gen Eimeria (92.8%). The high percentage found in Greece is
protozoa
h th
in accordance with
the results of previous similar studies. In the Czech Republic
and Slovakia coccidian oocyst were found in all (100%) of the examined European
ground squir
squirrels (Kviˇcerová 2008). In Bulgaria, Eimeria infection was found in
88.05% of S
S. citellus, with the most prevalent species being E. citelli (92.7%) and
E. callospermophili (66.6%) (Golemansky and Koshev 2009).
Levine and Ivens (1990) described 19 species of Eimeria in Spermophilus spp.
Of these species, E. callospermophili seems to be particularly common for the genus.
Apart from S. citellus, this parasite has been reported in Spermophilus xanthophrymnus
in Turkey (Çiçek et al. 2010), in Spermophilus richardsonii, Spermophilus townsendii,
Spermophilus lateralis and Spermophilus elegans in North America (Stanton et al.
1992; Seville and Stanton 1993; Wilber et al. 1994) and in Spermophilus parryii in
Alaska and Siberia (Seville et al. 2005).
125
130
135
140
145
150
155
160
165
Journal of Natural History 7
The identiﬁcation and taxonomy of Eimeria spp. has traditionally been based on
the morphology of the sporulated oocyst and the host where the examined species is
found (Levine and Ivens 1990). In the present study, apart from the three identiﬁed
Eimeria species, two more types of oocysts with different morphologies were detected
in 22 animals. Unfortunately, the number of morphological characteristics of Eimeria
spp. is limited whereas the number of species is large (Motriuk-Smith et al. 2011).
Development and application of molecular methods for the precise identiﬁcation of
these parasites are planned and would solve this problem.
Eimeria is a protozoan parasite with a direct life cycle that invades the epithelial
cells of the small and large intestine where it reproduces. There are no dat
data in the
literature about the pathogenicity of Eimeria in European ground squirrels. Ge
Generally,
coccidiosis in sciurids seems to cause few or no clinical signs (Joseph 1975; Coh
Cohn et al.
ulgaris) signs
1986; Sainsbury and Gurnell 1995). However, in red squirrels (Sciurus vulgaris)
nd eeven a fatal outcome
utco
such as loss of general condition, loss of appetite, diarrhoea and
have been reported (Pellerdy 1954, 1974).
nfecti n has not been
b
The immune response of Spermophilus spp. to Eimeria infection
clarbot the parasite
p
iﬁed. There is evidence that immunity depends on the species of both
and
d more than ad
the host. Moreover, the fact that juveniles are infected
adults due to ageer et al. 1994).
1994) However, Golemansky
developed immunity has not been proven (Wilber
th E. callospermophili
callospermop
and Koshev (2009) found heavier infection with
and E. cynomysis
(
in subadult and juvenile S. citellus and Seville and Stanton (1993)
found greater variety
an in adults. Taking into consideration,
of Eimeria species in juvenile S. richardsonii than
an heavy infectio
on the one hand, the high prevalencee and
infection with Eimeria spp. found in
er hand, the
t potential pathogenicity of the parasite
the present study, and on the other
ulations of
o S. citellus in Greece, it can be suggested
and the evidence of declining populations
resent a threat
threa for ground squirrels and a risk for the stathat this infection could represent
n the study area.
rea. More investigations towards this end are
bility of the populations in
p
currently underway, as thiss may affect populations
of the species that inhabit generally
n central
cen ral Greece.
favourable habitats in
ium is another
anoth
her genus of the subclass Coccidia. It has a direct life cycle
Cryptosporidium
omes infected by ingestion or inhalation of the oocysts found in food,
and the host becomes
e vironment in general. It is a parasite of the epithelial cells of the gaswater and thee environment
inal tract
trac (less comm
trointestinal
commonly of the respiratory tract) and is an important cause
trointestinal illness
ill
of gastrointestinal
for humans and animals (Dubey et al. 1990). Without having
ict host
host speciﬁcity, Cryptosporidium species are recognized to differ principally in
strict
hos range
rang but some genotypes are considered zoonotic (Hunter and Thompson
their host
2005).
t present study, Cryptosporidium spp. was found in 23.2% of the examined aniIn the
als. To
T the authors’ best knowledge, this is the ﬁrst report of this parasite in S. citellus.
mals.
eve 35.5% of the S. suslicus examined by direct immunoﬂuorescence assay in
However,
Poland were shedding Cryptosporidium oocysts (Kloch and Bajer 2012). Moreover,
Cryptosporidium parvum has been found in 16% of the Spermophilus beecheyi examined
in California (USA), where two different genotypes were circulating within a single
host population (Atwill et al. 2001). In a more recent survey, the parasite was found in
two more Spermophilus species (Spermophilus beldingi and Spermophilus lateralis) in
California and the phylogenetic analyses of the isolates revealed that Spermophilus
squirrels shed novel Cryptosporidium species (Pereira et al. 2010). As cross-species
transmission of Cryptosporidium spp. is a fact (Monis and Thompson 2003), molecular
170
175
180
185
190
195
200
205
210
215
8 A. Diakou et al.
characterization of isolates of this parasite would be useful in the investigation of the
possible transmission of the S. citellus genotypes to other animal species or humans.
Entamoeba is also a protozoan parasite with a direct life cycle. It parasitizes
the intestines of animals and humans and includes non-pathogenic and pathogenic
species. The reports of Entamoeba infection in sciurids are scant. The species reported
in Spermophilus are Entamoeba muris and Entamoeba citelli. The cystic form of
Entamoeba citelli (Becker 1926) is about 15 μm in diameter with eight nuclei and
a thick cystic wall. In the present study, the Entamoeba sp. cysts, found in morphologically good condition in very dry faeces, had all the characteristics described
at
for Entamoeba citelli. This parasite has been also reported in S. tridecemlineatus,
987
S. townsendii, S. beldingi and S. lateralis (Becker 1926; Davis 1969; Rickard 1987),
o the healt
and this is the ﬁrst report for S. citellus. The signiﬁcance of this parasite to
health
status of the sciurids is not known, as there are no relative published data.
Brachylaima is a trematode parasite of the intestines of mammalss an
and birds. It has
d in
termediate ho
an indirect life cycle with land snails and slugs as ﬁrst and second
intermediate
hosts
ously repor
(Yamaguti 1958; Butcher 2003). This parasitic trematode has been previ
previously
reported
tulus (López-D
arias et al.
from two species of the family Sciuridae, Atlantoxerus getulus
(López-Darias
ledge, this repo
2008) and Sciurus carolinensis (Kennedy 1988). To our knowledge,
report is a new
ection is rrelated
elate to the fact that
host record for Brachylaima spp. This trematode infection
008), which aactt as an intermediate
S. citellus often feeds on land snails (Coroiu et al. 2008),
host. This parasite was found only in Axios – eastern coast (site N
No. 3; Figure 2), where
ggs of this pa
seven of the 19 examined animals (36.8%) excreted eggs
parasite in their faeces.
logy of the parasite
arasite iis beyond the aim of the
Although the investigation of the epizootiology
n was limited
limi
present study, the fact that this infection
to only one population is interesting. This isolated occurrence could bee due to incidental infection of the particular
ransmiss on b
population, failure of infection transmission
because of short dispersal movements,
bers of snail
and/or predation of higher numbers
snails acti
acting as intermediate hosts (Helicidae)
ampling sites. Since the species B. criibi has been incicompared with the rest of the sampling
mans, iin
n Australia (B
dentally identiﬁed in humans,
(Butcher and Grove 2001), its presence in
ons relate
the site requires furtherr investigatio
investigations
related to human health.
The small number of helminth parasites found in S. citellus is consistent with the
rids, which in
general ﬁndings in sc
sciurids,
indicate that this family is parasitized at a lower level
than predicted (Moran
(Morand and Poulin 1998). Moreover, the relatively limited variation of
the parasiticc fauna found iin the present study could be attributed to the fact that our
tricted to non-i
study wass re
restricted
non-invasive faecal examination. Eventually, necropsies would
asitic species, but euthanasia is not an option, because of the vulnerable
reveal more pa
parasitic
us o
status
of S. ci
citellus.. R
Reports of helminth parasites in S. citellus are scant. Stefanov
et al. (2001) reported four helminth species, the cestodes Hymenolepis magaloon and
enia m
Ctenotaenia
marmotae and the nematodes Streptopharus kutasii and Trichostrongylus
is from Bulgaria.
colubriformis,
Despite the asocial behaviour of S. citellus, transmission of parasites is facilitated
between individuals due to the density of the colonies, which was relatively high in
all the sampled sites. Furthermore, the resistance of the reproductive elements of the
parasites to the environmental conditions is also a factor that contributes to the infection of the animals. In Greece, S. citellus inhabits low vegetation, relatively dry habitats
that are exposed to intense heat and solar radiation, especially during summer months.
Under such conditions, particularly resistant helminth eggs and protozoan cysts and
220
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225
230
235
240
245
250
255
260
Journal of Natural History 9
oocysts are vital for their survival. This may explain the very good condition of the
parasitic elements found in the present study, despite the dry faeces often examined.
This study provides the ﬁrst report on the parasitic fauna for S. citellus in Greece. 265
The high prevalence and in some cases heavy infection of the animals with Eimeria
should be further investigated to evaluate the signiﬁcance of the speciﬁc parasite to
the population health status and conservation. Close monitoring of the animals and
regular parasitological examinations would provide additional information towards
this end. The species of Eimeria and most probably Entamoeba reported in this study 270
are exclusive parasites of sciurids. In this way, no expected risk for domestic animals
ra
and public health exists. On the other hand, Cryptosporidium spp. and Brachylaima
pin of the
spp. may possibly affect other animals and humans. In every case, genotyping
parasites found in S. citellus would provide data for more accurate identiﬁcat
identiﬁcation and
ecies de
would facilitate the investigation of any probable genetic relation to species
derived 275
e, ssimilar investigations
gati
from other wild or domestic animals and humans. Furthermore,
on hosts that share the same habitats with S. citellus would be necessary for any conllus iin Greece an
sideration of actions plans for the conservation of S. citellus
and across
Europe.
Acknowledgements
We are particularly grateful to Col. K. Terpsidis and
nd Lt Col. G. Emmanouil for granting permission to work in the area of the C Army
Veterinary
yV
ry Hospital. We also express our gratitude
to the Managing Authority of the Axios
Delta for granting
os D
ti permission to work in the Area
under their supervision. Great thankss go to S. Vareltzidou,
L. Albanou and L.M. Rammou for
Va
working together in the ﬁeld. Financiall support for ﬁeldwork and laboratory analyses was partly
provided by the Aristotle University
ersity of Thessaloniki.
Thessa
280
285
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