R

Pw

CJa

b

a

ARRA

KCAGCTSC

1

amSiaWtw

G

h0

Veterinary Parasitology 233 (2017) 14–19

Contents lists available at ScienceDirect

Veterinary Parasitology

journa l homepage: www.e lsev ier .com/ locate /vetpar

esearch paper

rotozoan and helminth parasite fauna of free-living Croatian wildolves (Canis lupus) analyzed by scat collection

arlos Hermosilla a,∗, Sonja Kleinertz a, Liliana M.R. Silva a, Jörg Hirzmann a, Djuro Huber b,osip Kusak b, Anja Taubert a

Institute of Parasitology, Justus Liebig University Giessen, 35392 Giessen, GermanyFaculty of Veterinary Medicine, University of Zagreb, 10000 Zagreb, Croatia

r t i c l e i n f o

rticle history:eceived 5 September 2016eceived in revised form 8 November 2016ccepted 10 November 2016

eywords:anis lupusngiostrongylus vasorumiardiaryptosporidiumoxocaraarcocystisroatia

a b s t r a c t

The European wolf (Canis lupus) is a large carnivore species present in limited areas of Europe with sev-eral small populations still being considered as endangered. Wolves can be infected by a wide rangeof protozoan and metazoan parasites with some of them affecting free-living wolf health condition. Onthis account, an epidemiological survey was conducted to analyze the actual parasite fauna in Croatianwild wolves. In total, 400 individual faecal samples were collected during field studies on wolf ecol-ogy in the years 2002–2011. Parasite stages were identified by the sodium acetate acetic acid formalin(SAF)-technique, carbolfuchsin-stained faecal smears and Giardia/Cryptosporidium coproantigen-ELISAs.A subset of taeniid eggs-positive wolf samples was additionally analyzed by PCR and subsequent sequenc-ing to identify eggs on Echinococcus granulosus/E. multilocularis species level. In total 18 taxa of parasiteswere here detected. Sarcocystis spp. (19.1%) occurred most frequently in faecal samples, being followedby Capillaria spp. (16%), ancylostomatids (13.1%), Crenosoma vulpis (4.6%), Angiostrongylus vasorum (3.1%),Toxocara canis (2.8%), Hammondia/Neospora spp. (2.6 %), Cystoisospora ohioensis (2.1%), Giardia spp. (2.1%),Cystoisospora canis (1.8%), Cryptosporidium spp. (1.8%), Trichuris vulpis (1.5%), Taenia spp. (1.5%), Diphyl-lobothrium latum (1.5%), Strongyloides spp. (0.5%), Opisthorchis felineus (0.5%), Toxascaris leonina (0.3%),Mesocestoides litteratus (0.3%) and Alaria alata (0.3%). Some of the here identified parasites represent

relevant pathogens for wolves, circulating between these carnivorous definitive hosts and a varietyof mammalian intermediate hosts, e. g. Taenia spp. and Sarcocystis spp., while others are consideredexclusively pathogenic for canids (e.g. A. vasorum, C. vulpis, T. vulpis, Cystoisospora spp.). This study pro-vides first records on the occurrence of the two relevant anthropozoonotic parasites, Giardia spp. andCryptosporidium spp., in wild wolves from Croatia.

© 2016 Elsevier B.V. All rights reserved.

. Introduction

The European wolf (Canis lupus) used to be present in mostreas of Europe but during the 20th century it disappeared fromany European countries (Segovia et al., 2001; Stronen et al., 2011;

zafranska et al., 2010). Nowadays, the wolf is only present in lim-ted areas of Europe and respective populations are still considereds seriously endangered (Guerra et al., 2013; Segovia et al., 2001).

olves in Croatia belong to the large stable Dinaric-Balkan popula-ion of calculated 3900 individuals (Kaczensky et al., 2012). In 1995,hen a residual population of only 30 animals was left, wolves

∗ Corresponding author at: Institute of Parasitology, BFS, Schubertstr. 81, 35392iessen, Germany.

E-mail address: Carlos.R.Hermosilla@vetmed.uni-giessen.de (C. Hermosilla).

ttp://dx.doi.org/10.1016/j.vetpar.2016.11.011304-4017/© 2016 Elsevier B.V. All rights reserved.

became a protected species in Croatia. Nowadays its population hasslightly recovered from recent hunting and deforestation, and hasnow re-settled in the mountainous regions of Lika, Gorski Kotar andDalmatia (Frkovic and Huber, 1992; Kusak et al., 2005). Currently,the population ranges between 200 and 220 animals (Strbenacet al., 2010).

As large carnivores, wolves are natural hosts for a wide rangeof gastrointestinal parasites, some of them with zoonotic poten-tial such as Echinococcus spp. (Guerra et al., 2013; Ito et al., 2013;Schurer et al., 2014) and Toxocara canis (Segovia et al., 2001;Szafranska et al., 2010). So far, studies on wolf parasite infec-tions were performed in Nearctic (Choquette et al., 1973; Custer

and Pence, 1981; Holmes and Podesta, 1968; Messier et al., 1989;Samuel et al., 1978) and Palearctic regions (Craig and Craig, 2005;Guberti et al., 1993; Ito et al., 2013; Papadopoulos et al., 1997;Schurer et al., 2014; Shimalov and Shimalov, 2000) and para-

ry Par

soebcestfdwF2c(v

pBPIee(oS2ptbbra2

2

2

doNasJca(ocwMGpisl

2

Stf

C. Hermosilla et al. / Veterina

itoses are increasingly recognized for their profound influencesn individual or population health (Daszak et al., 2000; Hudsont al., 2006). Furthermore, parasite-host interactions are shapedy a multitude of factors, including host availability, parasiteommunity structure (Jolles et al., 2006; Telfer et al., 2010) andnvironmental conditions (Biek and Real, 2010). Moreover, para-itoses are increasingly associated with human-modified ecologicalransition zones (Despommier, 2007) and are of growing concernor isolated wilderness reserves (Stronen et al., 2011). Parasiticiseases can therefore severely reduce isolated populations evenithin protected geographical areas (see Despommier et al., 2006;

oreyt and Jessup, 1982; Leon-Vizcaino et al., 1999; Stronen et al.,011). As an example, mange due to Sarcoptes infestation is dis-ussed to have substantial impacts on wolf populations in SpainOleaga et al., 2011) as was also reported for red foxes (Vulpesulpes) (Al-Sabi et al., 2014; Nimmervoll et al., 2013).

Up to date, some investigations on wild wolf gastrointestinalarasites have been performed in other European countries, such aselarus (Shimalov and Shimalov, 2000), Spain (Segovia et al., 2001),ortugal (Guerra et al., 2013), Greece (Papadopoulos et al., 1997),

taly (Guberti et al., 1993), Poland (Kloch et al., 2005; Szafranskat al., 2010), Latvia (Bagrade et al., 2009) and Finland (Stronent al., 2013). Most investigations analyzed hunted or dead animalsEleni et al., 2014; Otranto et al., 2007, 2009; Segovia et al., 2001)r animals kept in captivity (Andre et al., 2010; Erdelyi et al., 2014;zafranska et al., 2010). So far, a survey on trichinellosis (Beck et al.,009) and on visceral leishmaniosis (Beck et al., 2008) have beenerformed in Croatian wolves. The present study aimed to iden-ify gastrointestinal parasitoses of free-ranging and alive wolvesy analyzing a high number of individual scat samples focusing onoth helminths and protozoans. Furthermore, we considered theole of these wild canids in the maintenance of synanthropic par-site cycles as discussed elsewhere (Guerra et al., 2013; Ito et al.,013; Schurer et al., 2014; Segovia et al., 2001).

. Material and methods

.1. Faecal samples and study area

In total 400 C. lupus scat samples were collected in 2002–2011uring field studies on wolf ecology and behavior in the mountain-us areas of Risnjak, Sniezik, Krasno, Paklenica, Suho, Snjeznik andic (Gorski Kotar region, Croatia). All scat samples were immedi-tely fixed in 90% ethanol until further investigation. Fixed faecalamples were thereafter shipped to the Institute of Parasitology,ustus Liebig University Giessen, Germany, for further coproscopi-al analysis. Coproscopical analyses included the standard sodiumcetate acetic acid formalin (SAF) technique applying ethyl acetateYang and Scholten, 1977; Young et al., 1979) for the detectionf parasite eggs, larvae, cysts, sporocysts and oocysts. In addition,arbol-fuchsin- stained faecal smears according to Heine (1982)ere carried out for the detection of Cryptosporidium spp. oocysts.oreover, commercial coproantigen-ELISAs for the detection of

iardia and Cryptosporidium infections (ProSpecT®

, Oxoid) wereerformed. The total subset of samples being microscopically pos-

tive for taeniid eggs (n = 7) was further analyzed by PCR andequencing in order to identify taeniid eggs to Echinococcus granu-osus/E. multilocularis species level.

.2. DNA extraction from faecal samples

DNA was extracted from faecal samples using the QIAamp DNAtool Mini kit (Qiagen, Germany) following glass bead homogeniza-ion as described by Nunes et al. (2006). 1 g of ethanol-preservedaecal samples was weighted into a 15 ml plastic tube, equilibrated

asitology 233 (2017) 14–19 15

in phosphate buffered saline at 4 ◦C overnight, centrifuged andresuspended in ASL extraction buffer using a glass rod. Then ∼30glass beads of 4 mm diameter (Carl Roth, Germany) were added. Thesample was homogenized by horizontal vortexing (Vortex Genie 2equiped with a 13000-V1-15 adapter, MO BIO Labs) and incubatedat 70 ◦C for 15 min. 2 ml of this homogenate were transferred to areaction tube, incubated at 95 ◦C for 10 min and after full speedcentrifugation (8000 x g, 10 min), and 1.2 ml of the supernatantwas further processed according to the manufacturers protocol byadding the inhibitor tablet and extracting the DNA.

2.3. Polymerase chain reaction and sequencing

For the molecular characterization of taeniid egg-positivesamples, nested PCRs being specific for the mitochondrial12S rDNA of E. multilocularis and E. granulosus sensu strictowere performed according to Dinkel et al. (1998, 2011) andStefanic et al. (2004) with minor modifications. For the first,cyclophyllid-specific PCR a revised P60.short.for (according tovon Nickisch-Rosenegk et al., 1999; Dinkel et al., 1998) for-ward primer (EmgrrnSs: 5′- TGACAGGGATTAGATACCC-3′) wasused in combination with the reverse primer P375.short.rev (5′-TGACGGGCGGTGTGTACC-3′; Dinkel et al., 2011). For the specificnested PCRs we used the following primer combinations: E. mul-tilocularis – Em-nest for (5′-GTGAGTGATTCTTGTTAGGGGAAGA-3′,Stieger et al., 2002; Dyachenko et al., 2008) and Pnest.rev. (5′-ACAATACCATATTACAACAATATTCCTATC-3′, Dinkel et al., 1998); E.granulosus – Eg1f (5′- CATTAATGTATTTTGTAAAGTTG-3′) and Eg1r(5′- CACATCATCTTACAATAACACC-3′, both Stefanic et al., 2004).The PCR reaction was performed in a 25 �l volume containing2.5 �l copro-DNA, 5 �l 5x Hot FIREPol Blend master mix, 0.5 �l7.5 mM MgCl2 (Solis BioDyne, Tartu, Estonia), 0.25 �l 10 mM BSAand 0.5 �l of each 10 �M primer. Amplification conditions for thefirst PCR were 15′ 95 ◦C initial denaturation, followed by 35 cyclesof 15′′ 95 ◦C, 30′′ 58 ◦C and 30′′ 72 ◦C and a final extension of5′ 72 ◦C. For the nested PCRs 1 �l of the first PCR was used at62 ◦C annealing for E. multilocularis and 58 ◦C for E. granulosus.For a further characterization of Echinococcus-negative samples,a semi-nested cyclophyllid PCR with the forward primer Cest5(5′-GCGGTGTGTACMTGAGCTAAAC-3′, Trachsel et al., 2007) andP375.short.rev was run with annealing at 58 ◦C. PCR ampliconswere gel-purified and sent to a commercial service (LGC Genomics,Berlin, Germany) for direct sequencing. Sequences were analyzedby BLAST search of the GenBank database.

3. Results

3.1. Wolves

A total of 400 individual wolf faecal samples were collected dur-ing the years 2002–2011. The spatial analysis of the tracking dataand the sample distribution revealed the presence of approximal-tely 48 packs within the area of investigation. The wolf pack rangeswere estimated by telemetric analyses within the Gorski Kotar andDalmatia area (Kusak et al., 2005). Pack territories consisted mainlyof mountainous mixed and coniferous forests, valleys, meadowsand villages. The total wolf population was estimated 220 in theGorski Kotar and Dalmatia regions (Strbenac et al., 2010). In theGorski Kotar area, roe deer (Capreolus capreolus), deer (Cervus ela-

phus), wild boars (Sus scrofa), hares (Lepus europaeus), and rabbits(Oryctolagus cuniculus) served as prey animals. In Dalmatia, most ofthe wolf diet referred to domestic animals (Huber, personal com-munication).

16 C. Hermosilla et al. / Veterinary Parasitology 233 (2017) 14–19

Table 1Prevalence (in percentage) of parasite species found in wild wolves (Canis lupus) faecal samples from Croatia (n = 400; years 2002–2011), and technique applied.

Groups Parasite species Prevalence (%) Technique

Protozoa Sarcocystis spp. 19.1 SAFHammondia/Neospora spp. 2.6 SAFCystoisospora ohioensis 2.1 SAFCystoisospora canis 1.8 SAFGiardia spp. 2.1 SAF/coproELISACryptosporidium spp. 1.8 coproELISA

Metazoa Capillaria spp. 16.0 SAFAncylostoma/Uncinaria spp. 13.1 SAFCrenosoma vulpis 4.6 SAFAngiostrongylus vasorum 3.1 SAFToxocara canis 2.8 SAFTrichuris vulpis 1.5 SAFTaenia spp. 1.5 SAF/PCRDiphyllobothrium latum 1.5 SAFMesocestoides litteratus 0.3 SAFOpisthorchis felineus 0.5 SAFToxascaris leonina 0.5 SAF

3

nlfCws(msltr0pseralsvot

3

siHCwtpd2ttS

Alaria alata

.2. Helminth species diversity

In total, twelve helminth species were recorded, with eightematode species, at least two cestode species (Diphyllobothrium

atum and Taenia spp.), and two trematode species (Opisthorchiselineus and Alaria alata) (Table 1). Within the nematode parasites,apillaria spp. infections (16%) were most frequently diagnosed inolf samples, being followed by ancylostomatids (13.1%), Creno-

oma vulpis (4.6%), Angiostrongylus vasorum (3.1%), Toxocara canis2.8%), Strongyloides spp. (0.5%) and Toxascaris leonina (0.3%). The

ost frequent cestode stages in wolf scat samples were Taeniapp. (1.5%) and Diphyllobothrium latum (1.5%) eggs. Mesocestoidesitteratus DNA was found with a very low prevalence (0.3%). Allrematode species, namely O. felineus and A. alata, were only spo-adically detected in wolf faeces, i. e. at low prevalences of 0.5% and.3%, respectively. A complete list of the detected parasite species isresented in Table 1. Furthermore, illustrations of selected parasitetages are depicted in Fig. 1. Overall, no acanthocephalan parasiteggs were found in wolf samples. None of the helminth specieseported here can be considered as a core species (prevalence > 50%)nd only two nematode species, i. e. Capillaria spp. and ancy-ostomatids, are the component species (>10% prevalence) of thetudy. Overall, three snail-borne parasitic infections (A. vasorum, C.ulpis, A. alata) and two fish-borne diseases (diphyllobothriosis andpisthorchiosis) were here identified, reinforcing the wolf alimen-ary flexibility and its adaptation capacities to diverse ecosystems.

.3. Protozoan species diversity

In contrast to helminths, the diversity of protozoan para-ites was less pronounced since only six species were foundn total. Thus, five apicomplexan parasites (Sarcocystis spp.,ammmondia/Neospora spp., Cystoisospora ohioensis, C. canis andryptosporidium spp.) and one metamonad species (Giardia spp.)ere diagnosed in wolf samples. Sarcocystis spp. infections were

he most prevalent (19.1%) ones, and revealed as the most prevalentarasites of the entire study, being followed by the Hammon-ia/Neospora complex (2.6%) and C. ohioensis and Giardia spp. (both

.1%). The lowest prevalences were detected for C. canis and Cryp-osporidium spp. (1.8%). Comparable to helminth infections, none ofhe protozoan parasites represented a core species and exclusivelyarcocystis spp. was a component species (>10%) in this study.

0.3 SAF

3.4. Molecular analysis of faecal samples containing taeniid eggs

3.4.1. Molecular analysis of taeniid eggsAll wolf samples containing taeniid eggs (n = 7) were analyzed

by specific copro-PCRs and molecular sequencing of the ampliconsto clarify from which species (e.g. Echinococcus spp. or Taenia spp.)they originated. PCR analyses revealed that all faecal samples werenegative for Echinococcus.

Further analysis using a semi-nested cestode universal PCR fol-lowed by direct sequencing of the amplicons identified three ofthe seven samples as T. hydatigena and two samples as T. multi-ceps. From the remaining two samples taeniid DNA could not beamplified but one contained DNA of Mesocestoides litteratus.

4. Discussion

Endoparasites of wolves have been investigated in most Euro-pean countries with endemic wild wolf populations (Szafranskaet al., 2010). However, the vast majority of these studies focusedon metazoan parasites and mainly analyzed necropsied animals(Abdybekova and Torgerson, 2012; Bagrade et al., 2009; Cirovicet al., 2015; Craig and Craig, 2005; Guerra et al., 2013; Ito et al.,2013; Otranto et al., 2007; Segovia et al., 2001). Thus, reports onfree-living animals are scarce (Gori et al., 2015; Stronen et al., 2011;Szafranska et al., 2010).

So far 72 different parasite species were reported in wolves and93% of these helminth species were detected via animal necrop-sies (Craig and Craig, 2005). The helminthofauna of wolves consistsof at least 28 nematode species, 27 cestode species, 16 trema-tode species and one acantocephalan species (Szafranska et al.,2010). The repertoire of wolf nematodes identified in the currentstudy is in accordance to previous reports (Craig and Craig, 2005).Overall, Croatian wolves showed rather low levels of parasitic infec-tions. Capillaria spp. (16%) and ancylostomatids (13.1%) revealedas the two current component nematode species (>10%), which isin agreement with data from Poland (Szafranska et al., 2010). Therather low level of ancylostomatid prevalence is in accordance tostudies in Italy, Belarus and Latvia (Segovia et al., 2003; Shimalovand Shimalov, 2000; Bagrade et al., 2009) but contrasts Spanishwolves which showed a much higher seasonal prevalence of up to

71.4% (Segovia et al., 2001). However, the current data show ancy-lostomatids as endemic in the Gorski Kotar region. This may bebased on the variety of infection routes enhancing the chance ofinfection. However, it has to be kept in mind that ancylostomatids

C. Hermosilla et al. / Veterinary Parasitology 233 (2017) 14–19 17

F from fA a, (E) O

ataaEsCCerBire

ig. 1. Depicted illustration of protozoan and metazoan parasite stages identified

ncylostomatidae egg, (C) Angiostrongylus vasorum larva, (D) Crenosoma vulpis larv

lso infect dogs and are a cause of zoonosis thereby posing a cer-ain threat on domestic and human population in the investigatedrea. It is generally known that wolves and other related canidsre (final) hosts of different Capillaria species [C. aerophila (syn.ucoleus aerophilus), C. boehmi (syn. E. boehmi), C. plica (syn. Pear-onema plica), C. putorii (syn. Aonchotheca putorii), C. hepatica (syn.alodium hepatica)] and of T. vulpis (Bagrade et al., 2009; Craig andraig, 2005; Guardone et al., 2013; Segovia et al., 2001; Szafranskat al., 2010). The current prevalence of Capillaria spp. (16%) wasather high when compared to studies performed in Canada (0.6%;ryan et al., 2012) but low when compared to Latvian data report-

ng on prevalences of 36.4% and 41.4% for C. aerophila and C. plica,

espectively (Bagrade et al., 2009). Nonetheless, it should be consid-red that the passage of intestinal parasite stages, such as Capillaria

aecal samples of Croatian wolves (Canis lupus): (A) Sarcocystis spp. sporocysts, (B)pisthorchis felineus egg, and (F) Diphyllobothrium latum egg (scale bars 10 �m).

eggs, frequently found in diverse prey animals, might lead to falsepositive animals.

The ascarid nematodes T. canis and T. leonina are well-known tooccur worldwide with T. leonina representing the most prevalentnematode species (meta-prevalence 73.9%) in tundra wolf popu-lations (Craig and Craig, 2005). In the current study, T. canis, andT. leonina were rarely diagnosed with 2.8 and 0.3%, respectively.In the case of T. canis, this may be explained by the relativelylow proportion of pups (<6 months) contributing to the currentstudy. Another feasible explanation for low T. canis- and T. leonina-prevalences could be that sampled animals belonged to one groupthereby having similar parasite exposure leading to an underesti-

mated prevalence if samples were taken from few wolf territories.Overall, the low prevalence of these nematodes might reflect the

1 ry Par

gh

iwIrc2aWoreeoondAjwweInwCpw

tattiwbGDwo

odrw2daHlwcC

twCylrIret

8 C. Hermosilla et al. / Veterina

eneral good health condition of the wolves and their rather intactabitat within the mountainous regions of Croatia.

Besides intestinal nematodes we also diagnosed lungwormnfections (C. vulpis and A. vasorum) in Croatian wolves. Both lung-

orms are capable to induce mild to severe respiratory disease.n the case of A. vasorum additionally severe cardiovascular, neu-al, ophthalmic and even systemic diseases have been described inanids (Chapman et al., 2004; Morgan et al., 2005; Taubert et al.,009; Patel et al., 2014) due to its localization in the right heartnd pulmonary arteries (Morgan et al., 2005; Taubert et al., 2009;

illesen et al., 2008) and ectopic spreading of larvae into differentrgans (Kirk et al., 2014; Patel et al., 2014). So far, only few studieseport on A. vasorum infections in European wolves (Spain: Segoviat al., 2001; Belarus: Shimalov and Shimalov, 2000 and Italy: Elenit al., 2014) showing similar prevalences to the current study. Inrder to study precise lungworm nematode prevalences in wolves,ther coproscopical methods such as the Baermann funnel tech-ique would have been the gold standard method to be used, butue ethanol fixation this technique was not applicable. In addition,. vasorum infections of other wild carnivores, such as the golden

ackal (Takacs et al., 2014) and foxes (Rajkovic-Janje et al., 2002)ere recently described. Crenosoma vulpis infections in wolvesere reported at higher prevalences from Latvia (9.1%, Bagrade

t al., 2009), Belarus (7.7%, Shimalov and Shimalov, 2000) and theberian Peninsula (10,6%, Segovia et al., 2001). Both lungworms sig-ificantly rely on snails/slugs as intermediate hosts, nonethelessolves are also known to feed on invertebrates. In fact, 9% of the

roatian wolf diet in the current study consisted of insects (Kusak,ersonal communications). Therefore, it is feasible to assume thatolves would also ingest mollusks as protein source.

The prevalence of cestodes and trematodes was generally low inhe current study, which is in accordance to other reports (Shimalovnd Shimalov, 2000; Cirovic et al., 2015). Molecular analysis ofaeniid-positive samples revealed an absence of Echinococcus infec-ions and proved infections with Taenia hydatigena and T. multicepsn Croatian wolves. These results correspond to data of Serbian

olf carcasses with low taeniid prevalences (Cirovic et al., 2015)ut contrast to reports on other European countries (Italy: 33%,ori et al., 2015; Spain: 64%, Segovia et al., 2001). The diagnosis ofiphyllobothrium spp. and Opisthorchis spp. infections in Croatianolves furthermore indicated that their nutritional habits obvi-

usly include fish as prey.In contrast to helminths, little is known on the occurrence

f intestinal protozoan parasites in wild wolves. The most abun-ant parasites found in this study were Sarcocystis species (19.1%),eflecting that the wolves annual diet mainly relies on deer andild boar as reported elsewhere (Bryan et al., 2012; Darimont et al.,

008). The occurrence of Sarcocystis infections in red deer, falloweer and wild boars is generally high with S. cervi and S. miescheri-na representing the most frequent species in Europe (Drost, 1977;eydorn and Matuschka, 1981). Although the here described preva-

ence matches to the worldwide Sarcocystis spp. prevalence inolves (19%, Craig and Craig, 2005), it proved rather low when

ompared to data from Alberta/Southern Alaska (28.2%, Craig andraig, 2005) and coastal Canadian regions (43.7%, Bryan et al., 2012).

In contrast to Sarcocystis spp., Giardia and Cryptosporidium infec-ions were barely diagnosed in Croatian wolf samples, both ofhich bear a relevant zoonotic potential. Especially in the case of

ryptosporidium this may be explained by the low proportion ofoung pups contribution to the current scat samples. Low preva-ences for Cryptosporidium infections in wolves were recently alsoeported from Canada (Bryan et al., 2012; Stronen et al., 2011).

n contrast, the current prevalence of Giardia infections (2.1%)evealed lower than that of Canadian wolves (21.9% and 6.8%, Bryant al., 2012; Stronen et al., 2011). The differences may be based onhe immune status and age distribution of Croatian wolves, their

asitology 233 (2017) 14–19

habitats or ecological aspects as postulated elsewhere (Bryan et al.,2012). Bryan et al. (2012) detected exclusively zoonotic assem-blages of Giardia (A, B) but not dog-specific assemblages (C, D) inwolf samples. Wolf territory sizes differ considerably within differ-ent geographic areas. In consequence, wolf pack home ranges varyfrom 100 to 359 km2 with an average of 230 km2 (Segovia et al.,2003), depending on the animal numbers and prey availability.Therefore, wolf territories often overlap with managed forests andfarmlands in human neighborhood resulting in direct or indirectcontacts with domestic animals and humans. Since wolves marktheir hunting territories by scats and urine (Zub et al., 2003) andalso prey on domestic animals, parasite transmission might becomea real risk factor for public health and likewise for the wolf popu-lation health. Consistently, reports on natural Giardia spp.-and C.parvum-infections in wild wolves (Kloch et al., 2005; Stronen et al.,2011) proved horizontal transmission between humans, domesticanimals and wild mammals.

Overall, this study adds additional data to the current knowl-edge on parasites of wild wolves and calls for more research in thisfield especially for monitoring reasons and to evaluate the possibleimpact of some here described parasitoses on wild wolf populationhealth status.

Acknowledgements

We greatly acknowledge the Croatian Large Carnivore Teamand their co-financers EURONATUR, the Bernd Thies Foundationand the Croatian State Institute for Nature Protection. Further, wedeeply acknowledge Birgit Reinhardt, Agnes Mohr and ChristineHenrich (Institute of Parasitology, Justus Liebig University Giessen,Germany) for their technical support in coprological faecal samplepreparations as well as DNA extraction and Echinococcus spp.-PCRs.

References

Cirovic, D., Pavlovic, I., Penezic, A., 2015. Intestinal helminth parasites of the greywolf (Canis lupus L.) in Serbia. Acta Vet. Hung. 63, 189–198.

Strbenac, A., Kusak, J., Huber, Ð., Jeremic, J., Okovic, P., Majic-Skrbinsek, A., Vuksic,I., Katusic, L., Desnica, S., Gomercic, T., Biscan, A., Zec, D., Grubesic, M., 2010.Plan upravljanja vukom u Republici Hrvatskoj za razdoblje od 2010. do 2015.godine, Ministarstvo kulture i Drzavni zavod za zastitu prirode, Zagreb.

Abdybekova, A.M., Torgerson, P.R., 2012. Frequency distributions of helminths ofwolves in Kazakhstan. Vet. Parasitol. 184, 348–351.

Al-Sabi, M.N.S., Halasa, T., Kapel, C.M.O., 2014. Infections with cardiopulmonaryand intestinal helminths and sarcoptic mange in red foxes from two differentlocalities in Denmark. Acta Parasitol. 59, 98–107.

Andre, M.R., Adania, C.H., Teixeira, R.H., Silva, K.F., Jusi, M.M., Machado, S.T., deBortolli, C.P., Falcade, M., Sousa, L., Alegretti, S.M., Felippe, P.A., Machado, R.Z.,2010. Antibodies to Toxoplasma gondii and Neospora caninum in captiveneotropical and exotic wild canids and felids. J. Parasitol. 96, 1007–1009.

Bagrade, G., Kirjusina, M., Vismanis, K., Ozolins, J., 2009. Helminth parasites of thewolf Canis lupus from Latvia. J. Helminthol. 83, 63–68.

Beck, A., Beck, R., Kusak, J., Gudan, A., Martinkovic, F., Artukovic, B., Hohsteter, M.,Huber, D., Marinculic, A., Grabarevic, Z., 2008. A case of visceral leishmaniosisin a gray wolf (Canis lupus) from Croatia. J. Wildl. Dis. 44, 451–456.

Beck, R., Beck, A., Kusak, J., Mihaljevic, Z., Lucinger, S., Zivicnjak, T., Huber, D.,Gudan, A., Marinculic, A., 2009. Trichinellosis in wolves from Croatia. Vet.Parasitol. 159, 308–311.

Biek, R., Real, L.A., 2010. The landscape genetics of infectious disease emergenceand spread. Mol. Ecol. 19, 3515–3531.

Bryan, H.M., Darimont, C.T., Hill, J.E., Paquet, P.C., Thompson, R.C., Wagner, B., Smits,J.E., 2012. Seasonal and biogeographical patterns of gastrointestinal parasitesin large carnivores: wolves in a coastal archipelago. Parasitology 139, 781–790.

Chapman, P.S., Boag, A.K., Guitian, J., Boswood, A., 2004. Angiostrongylus vasoruminfection in 23 dogs (1999–2002). J. Small Anim. Pract. 45, 435–440.

Choquette, L.P., Gibson, G.G., Kuyt, E., Pearson, A.M., 1973. Helminths of wolves,Canis lupus L., in the Yukon and northwest territories. Can. J. Zool. 51,1087–1091.

Craig, H.L., Craig, P.S., 2005. Helminth parasites of wolves (Canis lupus): a specieslist and an analysis of published prevalence studies in nearctic and palaearcticpopulations. J. Helminthol. 79, 95–103.

Custer, J.W., Pence, D.B., 1981. Dirofilariasis in wild canids from the gulf coastalprairies of Texas and Louisiana, USA. Vet. Parasitol. 8, 71–82.

ry Par

D

D

D

D

D

D

D

D

E

E

F

F

G

G

G

G

H

H

H

H

I

J

K

K

K

K

L

M

M

N

R., 2003. Wolf pack territory marking in the Bialowieza primeval forest(Poland). Behaviour 140, 635–648.

von Nickisch-Rosenegk, M., Lucius, R., Loos-Frank, B., 1999. Contributions to the

C. Hermosilla et al. / Veterina

arimont, C.T., Paquet, P.C., Reimchen, T.E., 2008. Spawning salmon disrupt trophiccoupling between wolves and ungulate prey in coastal British Columbia. BMCEcol. 8, 14.

aszak, P., Cunningham, A.A., Hyatt, A.D., 2000. Emerging infectious diseases ofwildlife-threats to biodiversity and human health. Science 287, 443–449.

espommier, D., Ellis, B.R., Wilcox, B.A., 2006. The role of ecotones in emerginginfectious diseases. Ecohealth 3, 281–289.

espommier, D.D., 2007. Chemical trails and the parasites that follow them. Proc.Natl. Acad. Sci. U. S. A. 104, 1447–1448.

inkel, A., von Nickisch-Rosenegk, M., Bilger, B., Merli, M., Lucius, R., Romig, T.,1998. Detection of Echinococcus multilocularis in the definitive host:coprodiagnosis by PCR as an alternative to necropsy. J. Clin. Microbiol. 36,1871–1876.

inkel, A., Kern, S., Brinker, A., Oehme, R., Vaniscotte, A., Giraudoux, P.,Mackenstedt, U., Romig, T., 2011. A real-time multiplex-nested PCR system forcoprological diagnosis of Echinococcus multilocularis and host species.Parasitol. Res. 109, 493–498.

rost, S., 1977. [Sarcosporidia of the cloven-hoofed game. III. Sarcosporidia in reddeer and fallow deer]. Angew. Parasitol. 18, 219–225.

yachenko, V., Pantchev, N., Gawlowska, S., Vrhovec, M.G., Bauer, C., 2008.Echinococcus multilocularis infections in domestic dogs and cats fromGermany and other European countries. Vet. Parasitol. 157, 244–253.

leni, C., De Liberato, C., Azam, D., Morgan, E.R., Traversa, D., 2014. Angiostrongylusvasorum in wolves in Italy. Int. J. Parasitol. Parasites Wildl. 3, 12–14.

rdelyi, K., Mezosi, L., Vladov, S., Foldvari, G., 2014. Fatal acute babesiosis in captivegrey wolves (Canis lupus) due to Babesia canis. Ticks Tick Borne Dis. 5, 281–283.

oreyt, W.J., Jessup, D.A., 1982. Fatal pneumonia of bighorn sheep followingassociation with domestic sheep. J. Wildl. Dis. 18, 163–168.

rkovic, A., Huber, D., 1992. Wolves in Croatia: baseline data. In: Promberger, C.,Schröder, W. (Eds.), Wolves in Europe—Status and Perspectives. MunichWildlife Society, Ettal, Germany, pp. 67–69.

ori, F., Armua-Fernandez, M.T., Milanesi, P., Serafini, M., Magi, M., Deplazes, P.,Macchioni, F., 2015. The occurrence of taeniids of wolves in Liguria (northernItaly). Int. J. Parasitol. Parasites Wildl. 4, 252–255.

uardone, L., Deplazes, P., Macchioni, F., Magi, M., Mathis, A., 2013. Ribosomal andmitochondrial DNA analysis of Trichuridae nematodes of carnivores and smallmammals. Vet. Parasitol. 197, 364–369.

uberti, V., Stancampiano, L., Francisci, F., 1993. Intestinal helminth parasitecommunity in wolves (Canis lupus) in Italy. Parassitologia 35, 59–65.

uerra, D., Armua-Fernandez, M.T., Silva, M., Bravo, I., Santos, N., Deplazes, P.,Carvalho, L.M., 2013. Taeniid species of the Iberian wolf (Canis lupus signatus)in Portugal with special focus on Echinococcus spp. Int. J. Parasitol. ParasitesWildl. 2, 50–53.

eine, J., 1982. An easy technique for the demonstration of cryptosporidia in feces.Zbl Vet. Med. B 29, 324–327.

eydorn, A.O., Matuschka, F.R., 1981. [Final host specificity of Sarcocystis speciestransmitted by dogs (authors’s transl)]. Z. Parasitenkd. 66, 231–234.

olmes, J.C., Podesta, R., 1968. Helminths of wolves and coyotes from forestedregions of alberta. Can. J. Zool. 46, 1193-&.

udson, P.J., Cattadori, M., Boag, B., Dobson, A.P., 2006. Climate disruption andparasite-host dynamics: patterns and processes associated with warming andthe frequency of extreme climatic events. J. Helminthol. 80, 175–182.

to, A., Chuluunbaatar, G., Yanagida, T., Davaasuren, A., Sumiya, B., Asakawa, M., Ki,T., Nakaya, K., Davaajav, A., Dorjsuren, T., Nakao, M., Sako, Y., 2013.Echinococcus species from red foxes, corsac foxes, and wolves in Mongolia.Parasitology 140, 1648–1654.

olles, A.E., Etienne, R.S., Olff, H., 2006. Independent and competing disease risks:implications for host populations in variable environments. Am. Nat. 167,745–757.

aczensky, P., Chapron, G., von Arx, M., Huber, D., Andrén, H., Linnell, J., 2012.Status, management and distribution of large carnivores – bear, lynx, wolf &wolverine – in Europe. Document prepared for the European Commission(contract 070307/2012/629085/SER/B3).

irk, L., Limon, G., Guitian, F.J., Hermosilla, C., Fox, M.T., 2014. Angiostrongylusvasorum in UK mainland: a nationwide postal questionnaire survey ofveterinary practices. Vet. Rec. 175 (5), 118–123.

loch, A., Bednarska, M., Bajer, A., 2005. Intestinal macro- and microparasites ofwolves (Canis lupus L.) from north-eastern Poland recovered by coprologicalstudy. Ann. Agric. Environ. Med. 12, 237–245.

usak, J., Skrbinsek, A., Huber, D., 2005. Home ranges, movements, and activity ofwolves (Canis lupus) in the Dalmatian part of Dinarids, Croatia. Eur. J. Wildl.Res. 51, 254–262.

eon-Vizcaino, L., Ruiz de Ybanez, M.R., Cubero, M.J., Ortiz, J.M., Espinosa, J., Perez,L., Simon, M.A., Alonso, F., 1999. Sarcoptic mange in Spanish ibex from Spain. J.Wildl. Dis. 35, 647–659.

essier, F., Rau, M.E., Mcneill, M.A., 1989. Echinococcus granulosus (Cestoda,Taeniidae) infections and moose wolf population-dynamics in southwesternquebec. Can. J. Zool. 67, 216–219.

organ, E.R., Shaw, S.E., Brennan, S.F., De Waal, T.D., Jones, B.R., Mulcahy, G., 2005.Angiostrongylus vasorum: a real heartbreaker. Trends Parasitol. 21, 49–51.

immervoll, H., Hoby, S., Robert, N., Lommano, E., Welle, M., Ryser-Degiorgis, M.P.,2013. Pathology of sarcoptic mange in red foxes (Vulpes vulpes): macroscopicand histologic characterization of three disease stages. J. Wildl. Dis. 49, 91–102.

asitology 233 (2017) 14–19 19

Nunes, C.M., Lima, L.G.F., Manoel, C.S., Pereira, R.N., Nakano, M.M., Garcia, J.F., 2006.Fecal specimens preparation methods for PCR diagnosis of human taeniosis.Rev. Inst. Med. trop. S. Paulo 48, 45–47.

Oleaga, A., Casais, R., Balseiro, A., Espi, A., Llaneza, L., Hartasanchez, A., Gortazar, C.,2011. New techniques for an old disease: sarcoptic mange in the Iberian wolf.Vet. Parasitol. 181, 255–266.

Otranto, D., Cantacessi, C., Mallia, E., Lia, R.P., 2007. First report of Thelaziacallipaeda (Spirurida, Thelaziidae) in wolves in Italy. J. Wildl. Dis. 43, 508–511.

Otranto, D., Dantas-Torres, F., Mallia, E., DiGeronimo, P.M., Brianti, E., Testini, G.,Traversa, D., Lia, R.P., 2009. Thelazia callipaeda (Spirurida, Thelaziidae) in wildanimals: report of new host species and ecological implications. Vet. Parasitol.166, 262–267.

Papadopoulos, H., Himonas, C., Papazahariadou, M., AntoniadouSotiriadou, K.,1997. Helminths of foxes and other wild carnivores from rural areas in Greece.J. Helminthol. 71, 227–231.

Patel, Z., Gill, A.C., Fox, M.T., Hermosilla, C., Backeljau, T., Breugelmans, K., Keevash,E., McEwan, C., Aghazadeh, M., Elson-Riggins, J.G., 2014. Molecularindentification of novel intermediate host species of Angiostrongylus vasorumin Greater London. Parasitol. Res. 113, 4363–4369.

Rajkovic-Janje, R., Marinculic, A., Bosnic, S., Benic, M., Vinkovic, B., Mihaljevic, Z.,2002. Prevalence and seasonal distribution of helminth parasites in red foxes(Vulpes vulpes) from the Zagreb County (Croatia). Z. Jagdwiss. 48, 151–160.

Samuel, W.M., Ramalingam, S., Carbyn, L.N., 1978. Helminths in coyotes (Canislatrans Say) wolves (Canis lupus L.), and red foxes (Vulpes vulpes L.) ofsouthwestern Manitoba. Can. J. Zool. 56, 2614–2617.

Schurer, J.M., Gesy, K.M., Elkin, B.T., Jenkins, E.J., 2014. Echinococcus multilocularisand Echinococcus canadensis in wolves from western Canada. Parasitology 141,159–163.

Segovia, J.M., Torres, J., Miquel, J., Llaneza, L., Feliu, C., 2001. Helminths in the wolf,Canis lupus, from north-western Spain. J. Helminthol. 75, 183–192.

Segovia, J.M., Guerrero, R., Torres, J., Miquel, J., Feliu, C., 2003. Ecological analyses ofthe intestinal helminth communities of the wolf, Canis lupus, in Spain. FoliaParasitol. 50, 231–236.

Shimalov, V.V., Shimalov, V.T., 2000. Helminth fauna of the wolf (Canis lupuslinnaeus, 1758) in belorussian polesie. Parasitol. Res. 86, 163–164.

Stefanic, S., Shaikenov, B.S., Deplazes, P., Dinkel, A., Torgerson, P.R., Mathis, A.,2004. Polymerase chain reaction for detection of patent infections ofEchinococcus granulosus (sheep strain) in naturally infected dogs. Parasitol.Res. 92, 347–351.

Stieger, C., Hegglin, D., Schwarzenbach, G., Mathis, A., Deplazes, P., 2002. Spatialand temporal aspects of urban transmission of Echinococcus multilocularis.Parasitology 124, 631–640.

Stronen, A.V., Sallows, T., Forbes, G.J., Wagner, B., Paquet, P.C., 2011. Diseases andparasites in wolves of the Riding Mountain National Park region Manitoba.Can. J. Wildl. Dis. 47, 222–227.

Stronen, A.V., Jedrzejewska, B., Pertoldi, C., Demontis, D., Randi, E., Niedzialkowska,M., Pilot, M., Sidorovich, V.E., Dykyy, I., Kusak, J., Tsingarska, E., Kojola, I.,Karamanlidis, A.A., Ornicans, A., Lobkov, V.A., Dumenko, V., Czarnomska, S.D.,2013. North-South differentiation and a region of high diversity in Europeanwolves (Canis lupus). PLoS One 8, e76454.

Szafranska, E., Wasielewski, O., Bereszynski, A., 2010. A faecal analysis of helminthinfections in wild and captive wolves, Canis lupus L., in Poland. J. Helminthol.84, 415–419.

Takacs, A., Szabo, L., Juhasz, L., Takacs, A.A., Lanszki, J., Takacs, P.T., Heltai, M., 2014.Data on the parasitological status of golden jackal (Canis aureus L, 1758) inHungary. Acta Vet. Hung. 62, 33–41.

Taubert, A., Pantchev, N., Vrhovec, M.G., Bauer, C., Hermosilla, C., 2009. Lungworminfections (Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylusabstrusus) in dogs and cats in Germany and Denmark in 2003–2007. Vet.Parasitol. 159, 175–180.

Telfer, S., Lambin, X., Birtles, R., Beldomenico, P., Burthe, S., Paterson, S., Begon, M.,2010. Species interactions in a parasite community drive infection risk in awildlife population. Science 330, 243–246.

Trachsel, D., Deplazes, P., Mathis, A., 2007. Identification of taeniid eggs in thefaeces from carnivores based on multiplex PCR using targets in mitochondrialDNA. Parasitology 134, 911–920.

Willesen, J., Bjornvad, C., Koch, J., 2008. Acute haemoabdomen associated withAngiostrongylus vasorum infection in a dog: a case report. Ir. Vet J. 61, 591–593.

Yang, J., Scholten, T., 1977. A fixative for intestinal parasites permitting the use ofconcentration and permanent staining procedures. Am. J. Clin. Pathol. 67,300–304.

Young, K.H., Bullock, S.L., Melvin, D.M., Spruill, C.L., 1979. Ethyl acetate as asubstitute for diethyl ether in the formalin-ether sedimentation technique. J.Clin. Microbiol. 10, 852–853.

Zub, K., Theuerkauf, J., Jedrzejewski, W., Jedrzejewska, B., Schmidt, K., Kowalczyk,

phylogeny of the Cyclophyllidea (Cestoda) inferred from mitochondrial 12SrDNA. J. Mol. Evol. 48, 586–596.