DOI 10.1007/s00436-016-5006-5 ORIGINAL PAPER Occurrence of Mesocestoides canislagopodis (Rudolphi, 1810) (Krabbe, 1865) in mammals and birds in Iceland and its molecular discrimination within the Mesocestoides species complex Karl Skirnisson 1 & Damien Jouet 2 & Hubert Ferté 2 & Ólafur K. Nielsen 3 Received: 27 January 2016 /Accepted: 10 March 2016 # Springer-Verlag Berlin Heidelberg 2016 Abstract ThelifecycleofMesocestoides tapeworms (Cestoda: Cyclophyllidea: Mesocestoididae) requires three hosts. The first intermediate host is unknown but believed to be an arthropod. The second intermediate host is a vertebrate. The primary definitive host is a carnivore mammal, or a bird of prey, that eats the tetrathyridium-infected second intermediate host. One representative of the genus, Mesocestoides canislagopodis, has been reported from Iceland. It is common in the arctic fox (Vulpes lagopus) and has also been detected in domestic dogs (Canis familiaris) and cats (Felis domestica). Recently, scolices of a non-maturing Mesocestoides sp. have also been detected in gyrfalcon (Falco rusticolus) intestines, and tetrathyridia in the body cavity of rock ptarmigan (Lagopus muta). We examined the taxonomic relationship of Mesocestoides from arctic fox, gyrfalcon, and rock ptarmigan using molecular methods, both at the generic level (D1 domain LSU ribosomal DNA) and at the specific level (cytochrome c oxidase subunit I (COI) and 12S mitochondrial DNA). All stages belonged to Mesocestoides canislagopodis. Phylogenetic analysis of the combined 12S-COI at the specific level confirmed that M. canislagopodis forms a distinct clade, well separated from three other recognized representatives of the genus, M. litteratus, M. lineatus, and M. corti/ vogae. This is the first molecular description of this species. The rock ptarmigan is a new second intermediate host record, and the gyrfalcon a new primary definitive host record. However, the adult stage seemed not to be able to mature in the gyrfalcon, and successful development is probably restricted to mammalian hosts. Keywords Iceland. Mesocestoides canislagopodis. Lagopus muta. Falco rusticolus. Vulpes lagopus. Tetrathyridia. Molecular biology * Karl Skirnisson karlsk@hi.is Introduction 1 2 3 Damien Jouet damien.jouet@univ-reims.fr Hubert Ferté hubert.ferte@univ-reims.fr Ólafur K. Nielsen okn@ni.is Laboratory of Parasitology, Institute for Experimental Pathology, University of Iceland, Keldur, IS-112 Reykjavík, Iceland EA4688 «Vecpar», UFR de Pharmacie, Université de Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51096 Reims Cedex, France Icelandic Institute of Natural History, Urriðaholtsstræti 6-8, 210 Garðabær, Iceland Adult Mesocestoides tapeworms (Cestoda: Cyclophyllidea: Mesocestoididae) are found in placental mammals and birds worldwide, with the exception of Australia (Padgett et al. 2013). The life cycle is assumed to require three hosts. The first intermediate host is unknown, but coprophagous arthropods are believed to play this role. Second intermediate hosts are insectivorous vertebrates (mammals, birds, amphibians, and reptiles) harboring a metacestode stage (tetrathyrida) in the body cavity. The adult tapeworm primarily parasitizes carnivorous mammals (canids, felids, and mustelids) that prey upon infected second intermediate hosts; humans and birds rarely act as definitive hosts (McAllister and Conn 1990, Loos-Frank 1991; Rausch 1994; Sato et al. 1999; Padgett and Boyce 2004; Padgett et al. 2005; Conn et al. 2011;
Gallas and Silveira 2011; Cho et al. 2013; Tokiwa et al. 2014). Peritoneal cestodiasis, caused by proliferative Mesocestoides tetrathyridia, is reported from dogs (Canis familiaris), cats (Felis domestica), and primates, indicating that some definitive hosts can also serve as intermediate hosts (reviewed by Padgett et al. 2013). Mesocestoides descriptions have traditionally been based on vague morphological characters and the identity of intermediate and definitive hosts. This was done despite of known species-specific morphological variability, lack of host specificity, and a wide geographic range. As a result, confusion and synonymies are common within the genus. Yamaguti (1959) recognized 27 species. Decades later, Chertkova and Kosupko (1978) considered 12 species to be valid and 11 as species of doubtful identity (species inquirendae). In Europe, Yanchev (1986) reported seven Mesocestoides species; at present, 12 species are listed in the Fauna Europea database (www.faunaeur.org). Molecular tools have significantly helped to identify Mesocestoides species, both by using ordinal and generic markers such as the 28S (D1 D3), 18S, and the ITS2 of the ribosomal DNA, or more specific domains such as cytochrome c oxidase 1 (COI) and 12S of the mitochondrial DNA (Bowles and McManus 1994; Nakao et al. 2000; Dinkeletal.2011; Eleni et al. 2007; Hrčkova et al. 2011; Littlewood et al. 1999, 2008; Padgett et al. 2013; Waeschenbach et al. 2007; Jabbar et al. 2012; Zalesny and Hildebrand 2012). Unfortunately, the use of these new tools has also led to submission of haplotypes without proper morphological characters for identifications (Foronda et al. 2007; Padgett et al. 2005; Padgett et al. 2013). To date, only three representatives of the genus Mesocestoides litteratus (Batsch, 1786), M. lineatus (Goeze, 1782), and M. corti (Hoeppli, 1925)(= M. vogae (Etges 1991) can reliably be identified as valid species, using both morphological and molecular characters (Padgett and Boyce 2005; Gubány and Eszterbauer 1998; Nickisch-Rosenegk et al. 1999; Literák et al. 2006; Hrčkova et al. 2011). In the early 1860s, an extensive examination was done in Iceland on a widespread and severe echinococcosis problem (Krabbe 1865). During these studies, Krabbe (1865) detected a previously unknown tapeworm in three definitive hosts: the arctic fox Vulpes lagopus, domestic dogs, and domestic cats. Krabbe (1865) described the new species as Taenia Canis lagopodis Rudolphi, 1810, but was not aware that a few years earlier, a new genus, Mesocestoides (Valliant 1863), had been described for cestodes lacking rostellar hooks, with genital organs along the midline of the proglottids, a distinct parauterine organ, and bipartite vitelline glands (Loos-Frank et al. 1992; Rausch1994). A century later, Baer (1962) named this tapeworm Mesocestoides litteratus (Batch, 1786) syn. T. canislagopodis Rudolphi, 1810. Some years later, Shults (1970) assigned the species to M. lineatus (Goeze, 1782). This taxonomical confusion encouraged Loos-Frank et al. (1992) to re-describe M. canislagopodis (Rudolphi, 1810) (Krabbe, 1865). Studies have shown that M. canislagopodis is very common in the arctic fox in Iceland (Loos-Frank et al. 1992; Skirnisson et al. 1993). In recent decades, the species has also occasionally been detected in domestic dogs (Skirnisson, unpublished). Clausen and Gudmundsson (1981) reported Mesocestoides sp. from an Icelandic gyrfalcon (Falco rusticolus); this was confirmed by Christensen et al. (2015) who found non-developing Mesocestoides sp. in gyrfalcons. Also, tetrathyridia of a Mesocestoides sp.havebeenfoundin the body cavity of rock ptarmigan (Lagopus muta) iniceland (Skirnisson et al., in press). In this study, we use both morphological and molecular tools to examine the taxonomic relationship of mature M. canislagopodis worms from the arctic fox, Mesocestoides sp. scolices from the gyrfalcon, and Mesocestoides sp. tetrathyridia from the body cavity of the rock ptarmigan. Also, we examined the taxonomical position of M. canislagopodis within the genus Mesocestoides. The geographical distribution is discussed, and remarks are made on the life cycle in Iceland. Material and methods Origin of the Mesocestoides material Mesocestoides material was obtained in Iceland from three hosts: 1. Dozens of adult worms (Fig. 1a) were isolated from the intestines of an adult arctic fox shot on 11 January 2012 on Audkuluheidi (65 13 N, 19 43 W) (IFEP Keldur accession number VL-12-01). 2. Scolices with undeveloped strobila were sampled from the small intestine of four gyrfalcons (Fig. 2), (Icelandic Institute of Natural History accession numbers FR-12-35, FR-12-15, FR-12-21, and FR-12-24) found dead or dying in Iceland in 2012 (Christensen et al. 2015). 3. Tetrathyridia (Fig. 1b) were sampled from the body cavity of an adult rock ptarmigan (Icelandic Institute of Natural History accession number LM-11-177), shot close to Lake Myvatn (65 37 N, 17 00 W) on 2 October 2011. Morphological identification of worms Adult worms were first examined fresh. After fixation and staining with chlorhydric carmine and mounting in Canada balsam (Fig. 3), the worms were studied by using Leica DMLB, Olympus BX50 microscope equipped for differential interference contrast (DIC) microscopy (Nomarski interference and phase contrast). Morphology of the tetrathyridia is described elsewhere (Skirnisson et al., in press). Measurements were made by using a calibrated eyepiece.
a b Fig. 1 a, b Light microscope photographs showing the morphology of Mesocestoides canislagopodis; a tetra-acetabulate scolex (no rostellum) and proglottids of the adult stage from arctic fox Vulpes lagopus in Iceland, each sucker with a longitudinal slit (arrow); b Type specimens were deposited at the Icelandic Institute of Natural History (accession number 36054). Photographs were made using a Nikon DS-Vi1 digital camera, alternatively attached to a Leica DMLB microscope or a Nikon SMZ 745T stereoscope. Molecular examination Fragments of worms and tetrathyridia were preserved in 95 % ethanol and frozen at 20 C for DNA analysis. Sequenced samples are listed in Table 1. After removing ethanol from the samples, DNA was extracted using the QIAmp DNA Mini Kit (Qiagen, Germany) following the manufacturer s instructions. During the first step (tissue lysis), parasites were crushed one by one using a piston pellet (Treff, Switzerland). Polymerase chain reactions (PCRs) and sequencing of the D1 domain of the 28S subunit of ribosomal DNA (rdna) were performed M. canislagopodis tetrathyridia (n = 13) taken alive in the body cavity of rock ptarmigan Lagopus muta in Iceland and allowed to relax for an hour in tap water. Scale bar 1mm under conditions described by Bowles and McManus (1994) with primers JB9 (5 -GCT GCA TTC ACA AAC ACC CCG ACT C-3 ) and JB10 (5 -GAT TAC CCG CTG AAC TTA AGC ATA-3 ). Two domains of the mitochondrial DNA (12S and COI) were amplified using the couple of primers Cest 60 (5 -TTA AGA TAT ATG TGG TAC AGG ATT AGA TAC CC-3 ) andcest75(5 -AAC CGA GGG TGA CGG GCG GTG TGT ACC-3 ) (Wirtherle et al.2007) and JB3 (5 -TTT TTT GGG CAT CCT GAG GTT TAT-3 ) and JB4.5 (5 -TAA AGA AAG AAC ATA ATG AAA ATG-3 ) (Bowles et al. 1995), respectively. PCR products were directly sequenced in both directions with the primers used for DNA amplification (Genoscreen, France). The sequences are deposited in GenBank under the accession numbers KT232137 to KT232152 (Table 1). Sequences were aligned using ClustalW that is included in MEGA version 5 software (Tamura et al. 2011) then were checked by eye. Sequences obtained from the different Mesocestoides stages in Iceland were compared with cestode sequences available in GenBank. The D1 domain of the rdna (212 bp) and the combined 12S and COI domains of the mitochondrial DNA (mdna; 689 bp) were used for the tree construction. Phylogenetic trees were constructed using the neighbor-joining (NJ), the maximum likelihood (ML), and minimum evolution (ME) methods, using the MEGA5 software. For each NJ, ML, and ME analyses, the most appropriate nucleotide substitution model was determined (HKY+G+ I), gaps were treated as missing data, and internal node support was assessed by bootstrapping over 500 replicates. Results Fig. 2 Light microscope photographs showing the morphology of 17 undeveloped Mesocestoides canislagopodis specimens from the small intestine of gyrfalcon Falco rusticolus (FR-12-35) in Iceland. This tapeworm does not mature in this avian host. Scale bar 1mm Morphology of parasites Arctic fox Strobili of gravid M. canislagopodis worms ranged 30 500 mm in length. The scolex is rectangular, with four
T CP 100 μm 100 μm C CP C CP U O V U PO 200 μm a b c Fig. 3 Morphology of three Mesocestoides canislagopodis proglottids isolated from arctic fox Vulpes lagopus in Iceland; a, b mature proglottids; c gravid proglottid. CP cirrus pouch, C cirrus, O ovaries, PO parauterine organ, T testes, U uterus, V vitellaria. Scale bars a, b 100 μm; c 200 μm suckers, each with a typical longitudinal slit. Shape and size of the scolices (scolex diameter 477 μm) and suckers (length = 193 μm; width = 204 μm) are similar with the description provided by Loos-Frank et al. (1992); rostellum is absent (Fig. 1a). Mature proglottids are square or slightly wider than long (Fig. 3a, b). Cirrus-pouch is oval, containing ejaculatory duct, and opens into the median genital atrium on the ventral surface. The testes are numerous (on average 62 per proglottid, range 53 74), in three dorsal rows, and extend throughout the length of the proglottid and join at the anterior margin. The ovary is bilobed and surrounded by two vitelline glands, both located near the posterior margin. The median uterus is ventral and slightly sinuous. Gravid proglottids are longer than wide and contain a fully developed oval parauterine organ filled with eggs (Fig. 3c). Gyrfalcon Dozens of M. canislagopodis specimens from four gyrfalcons were all immature with thin, short strobili that ranged 1 4 mm in length(fig. 2). Scolices were rectangular with a typical longitudinal slit as also seen in mature worms from the arctic fox (Fig. 1a), but size of scolices (diameter 357 μ m) and scolex sizes (length = 114 μ m; width = 157 μm) were smaller than the values of the original description. Proglottids were never mature or gravid. Rock ptarmigan Tetrathyridia from the body cavity of a subadult rock ptarmigan have the typical general morphology of genus Mesocestoides (Fig. 1b). Size and shape is variable and depend on the age of infection (Skirnisson et al., in press). Table 1 Isolates of Mesocestoides originating from Iceland used for molecular analysis GeneBank accession number Taxa Stage Host Location D1 COI 12S MES5 Adult Vulpes lagopus (VL-12-01) MES6 tetrathyridium Lagopus muta (LM-11-177) MES7 adult Falco rusticolus (FR-12-35) MES8 adult Falco rusticolus (FR-12-15) MES9 adult Falco rusticolus (FR-12-21) MES10 adult Falco rusticolus (FR-12-24) MES11 adult Vulpes lagopus (VL-12-01) MES12 adult Vulpes lagopus (VL-12-01) Iceland KT232137 KT232144 Iceland KT232138 KT232145 KT232151 Iceland KT232139 KT232146 Iceland KT232140 KT232147 Iceland KT232141 KT232148 Iceland KT232142 KT232149 Iceland KT232150 KT232152 Iceland KT232143
94 / 78 / 93 Rodentolepis asymmetrica Hymenolepis diminuta Hymenolepis hibernia Hymenolepis microstoma Hymenolepididae Dilepis undula 96 / 86 / 97 Monocercus arionis Dilepididae Anoplocephaloides variabilis 56 / 42 / 55 Bertiella anapolytica Phascolotaenia comani Progamotaenia villosa Anoplocephalidae Progamotaenia diaphana Progamotaenia bancrofti Raillietina tunetensis 83 / - / 78 Fuhrmannetta malakartis Raillietina sonini Davaineidae 89 / 90 / 89 Catenotaenia henttoneni 91 / 87 / 90 Skrjabinotaenia lobata Meggittina baeri Catenotaeniidae 81 / 75 / 82 Taenia (Hydatigera) taeniaeformis Taenia saginata Taeniidae 99 / 93 / 99 Taenia multiceps 100 / 96 / 100 Mesocestoides corti MES6 MES7 MES8 MES10 MES12 Mesocestoides sp. Mesocestoididae Amurotaenia deciduas Nippotaenia mogurndae Nippotaeniidae Molecular results 0.02 Fig. 4 Phylogenetic tree of Cyclophyllidea based on the D1 domain of the 28S rdna, constructed using the neighbor-joining method. The scale shows the number of nucleotide substitutions per site between DNA sequences. Amurotaenia deciduas (AF286932) and Nippotaenia Phylogenetic analyses of the D1 of the large ribosomal subunit, based on 32 sequences from 8 families of Cestoda, comprise 212 bp including gaps (Fig. 4). No variation was observed between sequence of Mesocestoides sp. (accession no. EF095263) isolated from striped field mouse (Apodemus agrarius) in Bulgaria and haplotypes from this study, corresponding to a tetrathyridium from rock ptarmigan, adult tapeworm from arctic fox, and scolices without strobila from gyrfalcon. Due to the conserved nature of this domain (only three variations between M. corti, Mesocestoides sp., and our sequences), the D1 domain was only used at family level to confirm the membership of our specimens to the family Mesocestoididae. This clade, formed by all sequences of Mesocestoides available, is well supported by bootstrap values. COI and 12S were used at the specific level. Molecular comparison of sequences obtained from this study with sequences of Mesocestoides spp. available from GenBank for these domains of the mdna (Table 2) confirmed that all mogurndae (AF286934) were chosen as outgroup. The node support is given in neighbor-joining, maximum likelihood, and minimum evolution bootstraps specimens isolated from arctic foxes (MES5, MES11, MES12), gyrfalcon (MES 7 10), and rock ptarmigan (MES6) in Iceland are homologous and differ from all currently deposited sequences. Molecular analysis of the mitochondrial 12S revealed different clades: (1) Mesocestoides sp. clade A from coyote (Canis latrans), dog, and the island fox (Urocyon littoralis) in North America; (2) Mesocestoides sp. clade B from coyote, mouse, and dog in North America, but also M. vogae (syn. M. corti) fromgreywolf(canis lupus) in Japan; (3) Mesocestoides sp. clade C from dog and island fox in North America; (4) Mesocestoides sp. from snow leopard (Uncia uncia) and red fox (Vulpes vulpes) in Mongolia; (5) M. corti from dog in Turkey; (6) M. lineatus from red fox in Slovakia; (7) M. lineatus from grey wolf, dog, and corsac fox (Vulpes corsac) in Mongolia; (8) all the sequences of M. litteratus from red fox (Slovakia) and dog (Turkey, Mongolia), M. leptothylacus and M. lineatus from red fox (Germany), and M. lineatus from striped field mouse (Bulgaria); and (9) sequences obtained from this study. For clades 5 and 7 and sequences of M. lineatus in clade
Table 2 Sequences of mitochondrial DNA (COI and 12S) of Mesocestoides spp. used for molecular comparison Species Author Host Country Stage Cox1 mdna 12S mdna M. litteratus Franssen et al. (unpublished) Vulpes vulpes Netherlands Adult KF751227-232 Hrčkova et al. 2011 Vulpes vulpes Slovakia Adult JF268502-525 JF268556-581 Zalesny and Hildebrand 2012 Myodes glareolus Poland Tetrathyridium JN088186 M. leptothylacus Nickisch-Rosenegk et al. 1999 Vulpes vulpes Germany Adult L49451 M. lineatus Hrčkova et al. 2011 Vulpes vulpes Slovakia Adult JF268500-01 JF268553-555 Narankhajid et al. (unpublished) Vulpes vulpes Mongolia Adult AB792715 AB787554 Canis familiaris Mongolia Adult AB792714 AB787553 Canis lupus Mongolia Adult AB792713 AB787552 Vulpes corsac Mongolia Adult AB792716 Nickisch-Rosenegk et al. 1999 Vulpes vulpes Germany Adult L49450 Wirtherleetal.2007 Canis familiaris Germany Tetrathyridium EF567417 M. vogae Nickisch-Rosenegk et al. 1999 Rattus norvegicus USA Adult L49448 Kashiide et al. (unpublished) Canis lupus familiaris Japan Tetrathyridium AB848991 AB848990 M. corti Aypak et al. (unpublished) Canis familiaris Turkey Tetrathyridium JN572111 Nakao et al. 2000 Japan Tetrathyridium AB033413 AB031363 Yildiz and Tong (unpublished) Canis familiaris Turkey Tetrathyridium HM011122 Mesocestoides sp. Jabbar et al. 2012 Felis domestica Italy Tetrathyridium JQ740884 Littlewood et al. 2008 Apodemus agrarius Bulgaria Tetrathyridium EU665469 EU665469 Narankhajid et al. (unpublished) Vulpes corsac Mongolia Adult AB792712 Canis lupus Mongolia Adult AB792720 Uncia uncia Mongolia Adult AB792719 AB787556 Vulpes vulpes Mongolia Adult AB792718 AB787555 Canis familiaris Mongolia Adult AB792717 Canis vulpes Mongolia Adult AB793741 Padgett et al. 2005 Urocyon littoralis USA Adult DQ104224-225 Adult DQ102762-765 Canis familiaris USA Adult DQ102741-742 Adult DQ102747-750 Adult DQ102753-755 Adult DQ102757-761 Mus musculus USA Tetrathyridium DQ102756 Canis latrans USA Adult DQ102739-740 Adult DQ102751-752 Peromyscus maniculatus USA Tetrathyridium DQ102746 Urocyon littoralis USA Adult DQ102743-745 Tamir et al. (unpublished) Canis familiaris Mongolia Eggs AB915950 Tokiwa et al. 2014 Simia sciureus Japan Tetrathyridium AB932596 AB908164 8, we assume that the names assigned to these sequences correspond to a misidentification. By analyzing the partial COI of mdna, among the Mesocestoididae, nine clades are recognized: (1) a clade formed by sequences of M. litteratus isolated from red fox in Slovakia; (2) M. lineatus from red fox in Slovakia; (3) M. lineatus from dog, grey wolf, red fox, and corsac fox in Mongolia; (4) M. corti/vogae in Japan and Mesocestoides sp. from striped field mouse in Bulgaria; (5) Mesocestoides sp. from snow leopard in Mongolia; (6) Mesocestoides sp. from red fox and grey wolf in Mongolia; (7) Mesocestoides sp. from cat in Italy; (8) Mesocestoides sp. from squirrel monkey (Simia sciureus) in Japan; and (9) a clade formed by our haplotypes. For clade 3, we assume that the name assigned to these sequences corresponds to a misidentification. Finally, phylogenetic analyses of the combined 12S-COI (Fig. 5), with sequences of the three species currently recognized molecularly and morphologically (M. litteratus, M. lineatus, and M. corti/vogae), confirm the membership of our sequences to a single clade, well supported by bootstrap
99/77/100 Mesocestoides litteratus 100/96/100 Mesocestoides corti (= vogae) 86/84/88 100/92/100 Mesocestoides lineatus MES6 Mesocestoides canislagopodis MES11 Nippotaenia chaenogobii 100/97/100 100/92/100 values for NJ, ML, and ME analyses. Intraspecific variability for our sequences is represented by two variations (99.7 % of homology), in comparison with pairwise genetic variability (9.2 to 14.8 %) comprised between Mesocestoides spp. These results confirm that our samples belong to a single species; this is the first molecular description of M. canislagopodis. Discussion Valid species 0.02 Fig. 5 Phylogenetic tree of Mesocestoides spp. based on the combined 12S-COI of the mdna, constructed using the neighbor-joining method. The scale shows the number of nucleotide substitutions per site between DNA sequences. Nippotaenia chaenogobii (JQ268550) was chosen as outgroup. The node support is given in neighbor-joining, maximum Formal taxonomic assignment of species within genus Mesocestoides is difficult as the range of morphometric measurements as well as geographic distribution markedly overlap among nominal species. Therefore, other methods, such as molecular characterization combined with morphological examinations, are needed (Hrčkova et al. 2011; Padgett et al. 2005). Currently, only three species are morphologically and molecularly recognized within the genus, two species mainly confined to Europe, M. litteratus (Batsch, 1786) and M. lineatus (Goeze, 1782), and M. corti Hoeppli, 1925(= M. vogae Etges, 1991) originally found in a lizard in California, North America. In Germany, Loos-Frank (1980) described M. leptothylacus and considered the name assigned to M. litteratus as nomen dubium. Recent molecular analyzes, however, have revealed that M. leptothylacus actually belongs to M. litteratus (Hrčkova et al. 2011). A number of other Mesocestoides spp. have also been isolated, but their morphological or molecular descriptions are still incomplete and therefore unclear as to whether they are new species or synonyms (Chertkova and Kosupko 1978; Hrčkova et al. 2011; Padgett et al. 2013). Morphological comparison Morphological studies of the genetically distinct M. lineatus and M. litteratus in Europe have shown that the two species likelihood, and minimum evolution bootstraps. M. litteratus and M. lineatus: sequences from Hrčkova et al. (2011); M. vogae (syn. corti): sequences from Kashiide et al. (unpublished); M. corti: sequences from Nakao et al. (2000) can be differentiated by discrete breaks in the ranges of some male and female reproductive characters (shape of the cirrus sack, length of cirrus, number of testes, position of ovaries, and vitellaria) but not on commonly examined tapeworm characters as morphology of scolex and strobila (Hrčkova et al. 2011). Morphometric comparison of M. lineatus, M. litteratus (Hrčkova et al. 2011), and M. canislagopodis revealed similar morphology and size measurements, and no clear differences of scolices and strobila were observed. Some differences, however, were observed in the form and size of the cirrus sack which appeared to be similar-sized in M. lineatus (on average 111 87 μm) and M. canislagopodis (148 90 μm), but thinner and longer in M. litteratus (198 57 μm). Numbers of testes were similar in M. litteratus (on average 56, range 50 75) and M. canislagopodis (62, 53 74), but noticeably fewer in M. lineatus (29, 25 38). Morphology of the parauterine organ also showed species-specific differences. M. litteratus has the longest parauterine organ (680 μm), M. canislagopodis is in the middle (608 μm), and M. lineatus has the shortest (478 μm). In conclusion, certain morphometrical values of M. canislagopodis more resemble those of M. lineatus;others are more close to M. litteratus, which supports our conclusion that reliable identifications must be performed with molecular analysis. Molecular analysis D1 domain Molecular analyses were conducted on the D1 domain of the large subunit (LSU) ribosomal DNA, a conserved domain which is informative at familial or ordinal levels for Cestoda (Bowles and McManus 1994; Lee et al. 2004; Olson et al. 2001), and at the generic level in particular for Mesocestoidinae Lühe, 1894, with only one genus described (Mesocestoides). Our results (Fig. 4) show a basal position of the Mesocestoididae clade in a phylogenetic tree of Cyclophyllidea. The position of this lineage, associated with particular life cycle and morphology of the
Mesocestoididae clade, is in favor of recognition at an ordinal rank (Hrčkova et al. 2011). The results also confirmed membership of samples isolated from arctic fox, gyrfalcon, and rock ptarmigan in Iceland, to genus Mesocestoides. Molecular analysis COI and 12S domains Two domains of the mitochondrial DNA (COI and 12S) were analyzed in order to compare at specific or population levels the sequences obtained in the present study with haplotypes already available in GenBank. Comparative analysis of the haplotypes obtained in Iceland, from arctic fox, gyrfalcon, and rock ptarmigan, revealed that all were conspecific and different from other already recognized species (M. litteratus, M. lineatus, and M. corti) as well as all other currently available haplotypes. M. canislagopodis therefore represents the fourth valid species of the genus. However, as no reliable species-specific morphological characters are known for M. canislagopodis, identification of the species has to be confirmed with molecular methods. Phylogenetic analyzes (Fig. 5) demonstrate M. canislagopodis as a sister group of M. lineatus from red foxes in Slovakia (Hrčkova et al. 2011), and a certain clade formed by different haplotypes isolated from dog, grey wolf, red fox, and corsac fox in Mongolia and molecularly described as M. lineatus (Narankhajid et al., unpublished). These haplotypes are probably a misidentification and should be regarded as an undescribed Mesocestoides sp. Is M. canislagopodis endemic to Iceland? The genus Mesocestoides is distributed worldwide except for Australia (Conn et al. 2011; Padgett et al. 2013). So far, M. canislagopodis has only been reported in Iceland (Loos-Frank et al. 1992; Skirnisson et al. 1993). Representatives of the genus are well known from Scandinavia and have for example been reported from red fox and raccoon dog in Denmark (Saeed et al. 2006; Al-Sabi et al. 2013, 2014) and from arctic foxes in Sweden (Meijer et al. 2011). In Greenland, Kapel and Nansen (1996) reported M. lineatus from arctic foxes, but these identifications have not been confirmed with molecular methods. Meijer et al. (2011) erroneously reported M. canislagopodis from the arctic fox in Svalbard, citing Stien et al. (2002), a reference on reindeer parasites, and not mentioning arctic fox parasites. This error was confirmed in an extensive study carried out on endoparasites of 353 arctic foxes from Svalbard where no Mesocestoides infections were recorded (Stien et al. 2010). The most likely source population for M. canislagopoides in Iceland is Greenland. The parasite might be carried either by stray arctic foxes, or potentially rock ptarmigan. Arctic foxes have frequently been observed on pack ice that irregularly forms an ice bridge between Iceland and Greenland (Braestrup 1941), and L. m. captus, the subspecies living in Northeast Greenland, has occasionally been found in Iceland (Guðmundsson 1972). Based on this scenario, we hypothesize that the closest relative of M. canislagopodis in Iceland will be found in the arctic fox in Greenland. On the life cycle in Iceland Mesocestoides tapeworms are generally believed to require three hosts to complete the life cycle. The first intermediate host for M. canislagopodis is still unknown. However, a very high infection prevalence and extensive infection of arctic foxes in Iceland (Loos-Frank et al. 1992; Skirnisson et al. 1993) suggest that the first intermediate should be widespread and abundant on the island. Previously, coprophagic arthropods (Foronda et al. 2007) and oribatid mites (Soldatova 1944) have been suggested to play this role (Loos-Frank 1987; Conn 1990; Rausch 1994). Ants have also been mentioned in this context (Padgett and Boyce 2005), but they do not occur in Iceland. As there are relatively few potential intermediate hosts available in Iceland (Ólafsson 1991), a systematic search for procercoids or other transitional metamorphosis stages or tetrathyridia in arthropods sampled in the vicinity of arctic fox dens might be successful in identifying the first intermediate host. If found, it is anticipated that these early post-hexacanth developmental stages would resemble the stages described during in vitro metamorphosis from oncosphere to tetrathyridium by Voge (1967) and Voge and Seidel (1968). Potential definitive hosts in Iceland Mesocestoides has a high prevalence in Icelandic gyrfalcons (27 %, n = 26 hosts), but no mature proglottids have been detected (Christensen et al. 2015). This suggests that birds are not playing a role as definitive hosts in maintaining the life cycle in Iceland. Furthermore, examination of intestines of 516 adult feral minks (Neovison vison), from various parts of Iceland in 1978 and 1987, never revealed Mesocestoides infections (Skirnisson, unpublished). Summing up the old findings of Krabbe (1865) and those obtained in recent decades suggests that mainly canids (arctic fox, dogs), but occasionally also felids (domestic cats), are able to act as definitive host of M. canislagopodis in Iceland. Tetrathyridia infections The present study confirmed that rock ptarmigan may act as intermediate host in the life cycle of M. canislagopodis. In general, limited knowledge is available on the occurrence of tetrathyridia in birds. Literák et al. (2004), using molecular methods, confirmed Mesocestoides tetrathyridia in the
common starling (Sturnus vulgaris) in the Czech Republic. Some decades earlier, Chertkova and Kosupko (1978) listed birds of seven orders, mostly Galliformes and Passeriformes, as intermediate hosts of Mesocestoides spp. in different European countries. Recently, Manios et al. (2002) reported tetrathyridium from the rock partridge Alectoris graeca in Greece and Millán et al. (2003) described Mesocestoides tetrathyridia from the body cavity of the red-legged partridge Alectoris rufa in Spain. Interestingly, the authors suggested that the low prevalence (2.7 %) might indicate that other vertebrates were far more important for the maintenance of the life cycle. Actually, this could also be the case in Iceland. In our survey on the parasites of the rock ptarmigan, even lower tetrathyridia infection prevalence has been reported (0.6 %, n = 1010 hosts (Skirnisson et al., in press). In Iceland, the arctic fox preys upon a number of birds other than rock ptarmigan (Hersteinsson 1993), species that have not been examined for the presence of tetrathyridia infections. Furthermore, the commonly occurring long-tailed field mouse Apodemus sylvaticus, an important prey species of the arctic fox in Iceland (Hersteinsson 1993), is also suspected to harbor tetrathyridia of M. canislagopodis. Studies performed in other European countries have frequently confirmed Mesocestoides tetrathyridia in rodents of the genus Apodemus, forexampleinpoland(zalesnyand Hildebrand 2012), Bulgaria (Literák et al. 2004), Sicily (Goüy de Bellocq et al. 2003), Spain (Conn et al. 2010), and Portugal (Eira et al. 2006). Potential hazard to humans Occasionally, humans serve as hosts to adult Mesocestoides tapeworms. To date, there have been at least 30 documented cases (reviewed by Padgett et al. 2013). Human infections have never been detected in Iceland (Skirnisson et al. 2003). However, as rock ptarmigan in Iceland represents an important food source for humans, the zoonotic potential of raw birds should be pointed out. The rock ptarmigan is a popular gamebird in Iceland, and for the period 2005 2012, the annual catch ranged between 35000 and 80000 birds (Environmental Agency of Iceland, http://www.ust. is/). Given the observed prevalence of M. canislagopodis tetrathyridia in rock ptarmigan (0.6 %, Skirnisson et al., in press), it is clear that some few hundred infected birds are being dressed and consumed annually. However, traditionally in Iceland, ptarmigan meat is not consumed raw but cooked, so that should minimize human infections. The main risk is probably for those who dress the game. One of the reported Mesocestoides human cases is from Greenland (Chandler 1949), but it was unknown how the infection was acquired. Conclusions In the present study, an integrative approach combining morphological and molecular analyses was used to describe M. canislagopodis which represents the fourth valid species of the genus Mesocestoides; the others are M. litteratus, M. lineatus, and M. corti/vogae. So far, M. canislagopodis has only been reported from Iceland. Definitive hosts for the species are canids (arctic fox, dogs) and occasionally felids (household cats); the adult stage seems not to be able to develop in the feral mink (mustelid) or reach maturity in an avian host as gyrfalcon. Rock ptarmigan was confirmed to host the tetrathyridia metacestode stage of the life cycle. Conditions to search for the unknown first intermediate hosts of the Mesocestoides life cycle should be favorable in Iceland as only one Mesocestoides species occurs and the arthropod fauna is impoverished. Acknowledgements The authors would like to thank Johann Evensen for providing the arctic fox for analysis. Nanna Daugbjerg Christensen helped with examining gyrfalcon parasites, and in the past 10 years more than 40 persons have assisted in the field and in the laboratory in sampling and examining rock ptarmigan. Bruce Conn commented on an early draft of the manuscript. Financial support was provided by the Research Fund of the University of Iceland and the Icelandic Research Fund (grant number 090207021), and the Icelandic Institute of Natural History. In France, financial support was provided by the PHC Jules Verne Program for French Icelandic scientific cooperation and Office National de la Chasse et de la Faune Sauvage (ONCFS). References Al-Sabi MN, Chriél M, Jensen TH, Enemark HL (2013) Endoparasites of the raccoon dog (Nyctereutes procyonoides)and the red fox(vulpes vulpes) in Denmark 2009 2012 a comparative study. Int J Parasitol Parasites Wildl 2:144 51 Al-Sabi MN, Halasa T, Kapel CM (2014) Infections with cardiopulmonary and intestinal helminths and sarcoptic mange in red foxes from two different localities in Denmark. Acta Parasitol 59:98 107 Baer JG (1962) Cestoda. The Zoology of Iceland. Vol. 2 (12). Ejnar Munksgaard, Copenhagen and Reykjavik, 63 pp Bowles J, McManus DP (1994) Genetic characterization of the Asian Taenia, a newly described taeniid cestode of humans. Am J Trop Med Hyg 50:33 44 Bowles J, Blair D, McManus DP (1995) A molecular phylogeny of the human schistosomes. Mol Phylogenet Evol 4:103 9 Braestrup FW (1941) A Study on the arctic fox in Greenland. Immigrations, fluctuations in numbers based mainly on trading statistics. Meddelelser om Grönland. Bd. 131. C.A. Reitzels Forlag. København Chandler AC (1949) Introduction to parasitology. John Wiley and Sons, New York Chertkova AN, Kosupko GA (1978) The suborder Mesocestoidata Skryabin, 1940. In: Ryzhikov KM (ed) Principles of Cestodology. Nauka, Moscow, pp 118 229 [In Russian] Christensen ND, Skirnisson K, Nielsen OK (2015) The parasite fauna of the gyrfalcon (Falco rusticolus) in Iceland. J Wildl Dis 51:929 33
Cho SH, Kim TS, Kong Y, Na BK, Sohn WM (2013) Tetrathyridia of Mesocestoides lineatus in Chinese snakes and their adults recovered from experimental animals. Korean J Parasitol 51:531 6 Clausen B, Gudmundsson F (1981) Causes of mortality among freeranging gyrfalcons in Iceland. J Wildl Dis 17:105 9 Conn DB (1990) The rarity of asexual reproduction among Mesocestoides tetrathyridia (Cestoda). J Parasitol 76:453 5 Conn DB, Galán-Puchades MT, Fuentes MV (2010) Interactions between anomalous excretory and tegumental epithelia in aberrant Mesocestoides tetrathyridia from Apodemus sylvaticus in Spain. Parasitol Res 106:1109 15 Conn DB, Galán-Puchades MT, Fuentes MV (2011) Normal and aberrant Mesocestoides tetrathyridia from Crocidura spp. (Soricimorpha) in Corsica and Spain. J Parasitol 97:915 9 Dinkel A, Kern S, Brinker A, Oehme R, Vaniscotte A, Giraudoux P, Mackenstedt U, Romig T (2011) A real-time multiplex-nested PCR system for coprological diagnosis of Echinococcus multilocularis and host species. Parasitol Res 109:493 8 Eira C, Torres J, Vingda J, Miquel J (2006) Ecological aspects influencing the helminth community of the wood mouse Apodemus sylvaticus in Dunas de Mira, Portugal. Acta Parasitol 51:300 8 Eleni C, Scaramozzino P, Busi M, Ingrosso S, D Amelio S, De Liberato C (2007) Proliferative peritoneal and pleural cestodiasis in a cat caused by metacestodes of Mesocestoides sp. Anatomohistopathological findings and genetic identification. Parasite 14:71 6 Etges FJ (1991) The proliferative tetrathyridium of Mesocestoides vogae sp. n. (Cestoda). J Helminthol Soc Wash 58:181 5 Foronda P, Perez Rivero A, Santana Morales MA, Kabdur A, Gonzalez AC, Quispe Ricalde MA, Feliu C, Valladares B (2007) First larval record of Mesocestoides in carnivora of Tenerife (Canary Islands). J Parasitol 93:138 42 Gallas M, Silveira EF (2011) Mesocestoides sp. (Eucestoda, Mesocestoididae) parasitizing four species of wild felines in Southern Brazil. Rev Bras Parasitol Vet 20:168 70 Gubány A, Eszterbauer E (1998) Morphological investigation of Mesocestoides (Cestoda, Mesocestoididae) species parasitizing Vulpes vulpes in Hungary. Miscellanea Zoologica Hungarica 12: 11 9 Guðmundsson F (1972) Grit as an indicator of the overseas origin of certain birds occurring in Iceland. IBIS 114:582 Goüy de Bellocq J, Sarà M, Casanova JS, Feliu C, Morand S (2003) A comparison of the structure of helminth communities in the woodmouse, Apodemus sylvaticus, on islands of the western Mediterranean and continental Europe. Parasitol Res 90:64 70 Hersteinsson P (1993) Tófan [The Arctic fox]. In: Hersteinsson P, Sigbjarnarson G (eds) Villt, íslensk spendýr. HÍN and Landvernd, Reykjavík, pp 15 48 [In Icelandic] Hoeppli RJC (1925) Mesocestoides corti, a new species of cestode from the mouse. J Parasitol 12:91 6 Hrčkova G, Miterpakova M, O Connor A, Snabel V, Olson PD (2011) Molecular and morphological circumscription of Mesocestoides tapeworms from red foxes (Vulpes vulpes) in central Europe. Parasitology 138:638 47 Jabbar A, Papini R, Ferrini N, Gasser RB (2012) Use of a molecular approach for the definitive diagnosis of proliferative larval mesocestoidiasis in a cat. Infect Genet Evol 12:1377 80 Kapel CM, Nansen P (1996) Gastrointestinal helminths of Arctic foxes (Alopex lagopus) from different bioclimatological regions in Greenland. J Parasitol 82:17 24 Krabbe H (1865) Helminthologiske undersøgelser i Denmark og paa Island med særlige hensyn til blæreormelidelser paa Island. Kongelige Danske Videnskabernes Selskabs Skrifter, 5. Række, naturvidensk. og mathem. afdeling, 7. Bind, 71pp [In Danish] Lee SU, Huh S, Sohn WM, Chai JY (2004) Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai. Korean J Parasitol 42:129 35 Literák I, Olson PD, Georgiev BB, Spakulova M (2004) First record of metacestodes of Mesocestoides sp. in the common starling (Sturnus vulgaris) in Europe, with and 18S rdna characterization of the isolate. Folia Parasitol 51:45 9 Literák I, Tenora F, Letkova V, Goldova M, Torres J, Olson PD (2006) Mesocestoides litteratus (Batsch, 1786) (Cestoda: Cyclophyllidea: Mesocestoididae) from the red fox: morphological and 18S rdna characterization of European isolates. Helminthologia 43:191 5 Littlewood DTJ, Rohde K, Bray RA, Herniou EA (1999) Phylogeny of the Platyhelminthes and the evolution of parasitism. Biol J Linn Soc 68:257 87 Littlewood DTJ, Waeschenbach A, Nikolov PN (2008) In search of mitochondrial markers for resolving the phylogeny of cyclophyllidean tapeworms (Platyhelminthes, Cestoda) a test study with Davaineidae. Acta Parasitol 53:133 44 Loos-Frank B (1980) Mesocestoides leptothylacus n. sp.und das nomenklaturische Problem in der Gattung Mesocestoides Vaillant, 1863 (Cestoda, Mesocestoididae). Tropenmedizin und Parasitologie 31:1 14 [In German] Loos-Frank B (1987) Shedding of gravid proglottids and destrobilation in experimental infections of foxes with Mesocestoides leptothylacus Loos-Frank, 1980 (Cestoda). J Helminthol 61:213 18 Loos-Frank B (1991) One or two intermediate hosts in the life cycle of Mesocestoides (Cyclophyllidea, Mesocestoididae)? Parasitol Res 77:726 28 Loos-Frank B, Skirnisson K, Eydal M (1992) Mesocestoides canislagopodis (Rudolphi, 1810) (Krabbe, 1865) (Cestoda: Mesocestoididae) from arctic foxes, Alopex lagopus (L.) in Iceland redescribed. Bull Scand Soc Parasitol 2:68 73 Manios N, Papazahariadou M, Frydas S, Papageorgiou N, Tsachalidis E, Georgopoulou J (2002) Tetrathyridium as a mortality factor of rock partridge (Alcetoris graeca gracea) in Central Greece. Z Jagdwiss 48:378 82 McAllister CT, Conn DB (1990) Occurrence of Mesocestoides sp. tetrathyridia (Cestoidea: Cyclophyllidea) in North American anurans (Amphibia). J Wildl Dis 26:540 43 Meijer T, Mattson R, Angerbjörn A, Osterman-Lind E, Fernández- Aguilar X, Gavier-Widén D (2011) Endoparasites in the endangered Fennoscandian population of arctic foxes (Vulpes lagopus). Eur J Wildl Res 57:923 7 Millán J, Gortazar C, Casanova JC (2003) First occurrence of Mesocestoides sp. in a bird, the red-legged partridge, Alectoris rufa, in Spain. Parasitol Res 90:80 1 Nakao M, Sako Y, Yokoyama N, Fukunaga M, Ito A (2000) Mitochondrial genetic code in cestodes. Mol Biochem Parasitol 111:415 24 Nickisch-Rosenegk M, Richard L, Loos-Frank B (1999) Contributions to the phylogeny of the Cyclophyllidea (Cestoda) inferred from mitochondrial 12S rdna. J Mol Evol 48:586 96 Ólafsson E (1991) Íslenskt skordýratal [Insects of Icleland]. Fjölrit Náttúrufræðistofnunar 17:1 69 [In Icelandic] Olson PD, Littlewood DT, Bray RA, Mariaux J (2001) Interrelationships and evolution of the tapeworms (Platyhelminthes: Cestoda). Mol Phylogenet Evol 19:443 67 Padgett KA, Boyce WM (2004) Life-history studies on two molecular strains of Mesocestoides (Cestoda: Mesocestoididae): identification of sylvatic hosts and infectivity of immature life stages. J Parasitol 90:108 13 Padgett KA, Boyce WM (2005) Ants as first intermediate hosts of Mesocestoides on San Miguel Island, USA. J Helminthol 79:67 73 Padgett KA, Nadler SA, Munson L, Sacks B, Boyce WM (2005) Systematics of Mesocestoides (Cestoda: Mesocestoididae): evaluation of molecular and morphological variation among isolates. J Parasitol 91:1435 43
Padgett KA, Crosbie PR, Boyce WM (2013) Mesocestoides. In: Liu D (ed) Molecular detection of human pathogens. CRC Press, Taylor and Francis Group, pp 277 85 Rausch RL (1994) Family Mesocestoididae Fuhrmann, 1907. In: Khalil LF, Jones A, Bray RA (eds) Keys to the cestode parasites of vertebrates. CAB International, Wallingford, pp 309 14 Saeed I, Maddox-Hyttel C, Monrad J, Kapel CM (2006) Helminths of red foxes (Vulpes vulpes) in Denmark. Vet Parasitol 139:168 79 Sato H, Ihama Y, Inaba T, Yagisawa M, Kamiya H (1999) Helminth fauna of carnivores distributed in north-western Tohoku, Japan, with special reference to Mesocestoides paucitesticulus and Brachylaima tokudai. JVetMedSci61:1339 42 Shults LM (1970) Mesocestoides kirby and M. lineatus: occurrence in Alaskan carnivores. Trans Am Microsc Soc 89:478 86 Skirnisson K, Eydal M, Gunnarsson E, Hersteinsson P (1993) Parasites of the arctic fox Alopex lagopus in Iceland. J Wildl Dis 29:440 46 Skirnisson K, Richter SH, Eydal M (2003) Prevalence of human parasites in Iceland: past and present status. In: Akkuffo H, Ljungström I, Linder E, Whalgren M (eds) Parasites of the colder climates. Taylor and Francis, London and New York, pp 34 44 Skirnisson K, Sigurðardóttir ÓG, Nielsen ÓK. Morphological characteristics of Mesocestoides canislagopodis (Krabbe 1865) tetrathyridia found in rock ptarmigan Lagopus muta in Iceland. Parasitol Res (in press) Stien A, Irvine RJ, Ropstad E, Halvorsent O, Langvatn R, Albon SD (2002) The impact of gastrointestinal nematodes on wild reindeer: experimental and cross-sectional studies. J Anim Ecol 71:937 45 Stien A, Voutilainen L, Haukisalmi V, Fuglei E, Mørk T, Yoccoz NG, Ims RA, Henttonen H (2010) Intestinal parasites of the Arctic fox in relation to the abundance and distribution of intermediate hosts. Parasitology 137:149 57 Soldatova AP (1944) A contribution to the study of the development cycle in cestoda Mesocestoides lineatus (Goeze, 1782), parasitic of carnivorous mammals. Dokl Akad Nauk USSR 45:310 12 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.molbiolevol28:2731 9 Tokiwa T, Taira K, Yamazaki M, Kashimura A, Une Y (2014) The first report of peritoneal tetrathyridiosis in squirrel monkey (Saimiri sciureus). Parasitol Int 63:705 7 Vaillant L (1863) Sur deux helminthes cestoïde de la Genette. Inst, Paris, 1 er Sect, Sc Math. Phys Nat 31:87 8 Voge M (1967) Development in vitro of Mesocestoides (Cestoda) from oncosphere to young tetrathyridium. J Parasitol 53:78 82 Voge M, Seidel JS (1968) Continuous growth in vitro of Mesocestoides (Cestoda) from oncosphere to fully developed tetrathyridium. J Parasitol 54:269 71 Waeschenbach A, Webster BL, Bray RA, Littlewood DT (2007) Added resolution among ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) with complete small and large subunit nuclear ribosomal RNA genes. Mol Phylogenet Evol 45:311 25 Wirtherle N, Wiemann A, Ottenjann M, Linzmann H, van der Grinten E, Kohn B, Gruber AD, Clausen PH (2007) First case of canine peritoneal larval cestodosis caused by Mesocestoides lineatus in Germany. Parasitol Int 56:317 20 Yamaguti S (1959) Systhema helminthum: the cestodes of vertebrates. Interscience Publishers Inc., New York Yanchev Y (1986) On the morphology, taxonomy and distribution of the species of the genus Mesocestoides Vailland, 1863 in Bulgaria. Khelmintologiya 21:45 65 [In Bulgarian] Zalesny G, Hildebrand J (2012) Molecular identification of Mesocestoides spp. from intermediate hosts (rodents) in central Europe (Poland). Parasitol Res 110:1055 61