Introduction

Genus Mesocestoides is a representative of the small cyclophyllidean family Mesocestoididae Fuhrmann, 1907 that is found parasitizing the small intestine of carnivores, including canids, felids, and mustelids and rarely in birds. The characteristic features of this family are: a life cycle with three hosts (in the genus Mesocestoides), a genital atrium located medioventrally, and a bipartite vitelline gland (Rausch 1994). The life cycle of cestodes from genus Mesocestoides is complex and requires two intermediate hosts. Cysticercoids are produced in the first intermediate host (oribatid mites), which when eaten by the second intermediate host (mainly rodents, but also other mammalian species, birds, reptiles, or amphibians) form tetrathyridia in the host’s body cavity. According to Chertkova and Kosupko (1978), tetrathyridia were reported in about 200 species of vertebrates.

Taxonomical identification of Mesocestoides species based on morphomertical data remains problematic. More than a decade of research have identified the most significant characteristics in species determination of adult tapeworms as the shape of the cirrus pouch, the position and number of testes, and the position of the female sex organs (Literak et al. 2006; Priemer 1982; Tenora 2005). In larval stages isolated from intermediate hosts, the identification to the species level is impossible. However, the presence of metacestodes of Mesocestoides spp. or Mesocestoides lineatus have been reported in the helminth fauna of rodents (Bajer et al. 2005; Behnke et al. 2001; Behnke et al. 2008; Conn et al. 2010).

Seven species of this genus were recorded (often sympatrically) in Europe (Chertkova and Kosupko 1978; Yanchev 1986). Studies by Literak et al. (2004, 2006), Tenora (2005), and Hrckova et al. (2011) of the taxonomic status of Mesocestoides parasitizing red foxes in Europe (Czech Republic, Slovak Republic, and Spain) showed that Mesocestoides litteratus is the dominant species in relation to M. lineatus. From the other hand, according to Fauna Europaea data, M. lineatus occurs in most European countries, outside of the Apennine Peninsula. However, more recent studies on wildlife and domestic carnivores do not confirm these results (Andras and Peter 2002; Segovia et al. 2003; Martinez-Carrasco et al. 2007; Krone et al. 2008; Magi et al. 2009). Additionally, almost nothing is known about the ranges of their intermediate and paratenic hosts in natural conditions (Literak et al. 2004).

Despite the rich history of nomenclatural evaluation of Mesocestoididae, the taxonomic status within genus Mesocestoides is still unclear. Species identification based on morphological features of tetrathyridia has proven problematic, if not impossible. Thus, the aim of our study was to identify a molecular characteristic of the isolates of Mesocestoides from the second intermediate hosts (rodents) based on nuclear and mitochondrial ribosomal DNA data.

Materials and methods

Collection of rodents was conducted between 2001 and 2009 in the Wroclaw vicinity, i.e., an irrigation field—51°09′38″ N/16°58′50″ E (WO) and recreation grounds—51°10′19″ N/16°57′02″ E (WR) and in the northeast part of Lower Silesia in Nature Reserve “Stawy Milickie”—51°31′56″ N/17°20′12″ E (RM).

We choose to analyze metacestodes isolated from the body cavity of two species of rodents from different sites (i.e., Apodemus agrarius, WO; A. agrarius, WR; Myodes glareolus, WR; and M. glareolus, RM). The total genomic DNA from tetrathyridia was extracted by the use of commercial kit (Qiagen DNeasy tissue kit) following manufacturer’s protocol. Before extraction, the tapeworm’s tissue was heated in order to evaporate preserved ethanol.

Amplification of partial sequence of 18S rDNA was made using primers WormA (5′-GCGAATGGCTCATTAAATCAG-3′)/1270R (5′-CCGTCAATTCCTTTAAGTTT-3′) (Littlewood and Olson 2001) in the following thermocycling conditions: 94°C/5 min—initial denaturation; 94°C/1 min, 52°C/1 min, and 72°C/2 min—40 cycles; 72°C/7 min—final extending (Literak et al. 2006). The PCR reaction (25 μl) was performed usingthe following PCR mix: 2 μl of genomic DNA, 10 mM Tris–HCl, 50 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP, 150 pmol of each primer, and two units of Taq polymerase (Qiagen). In addition, the same extracted genomic DNA of metacestodes from M. glareolus (RM) was used to amplify the fragment of mitochondrial 12S rDNA gene. The following primers were used 60. for (5′-TTAAGATATATGTGGTACAGGATTAGATACCC-3′) and 75 rev (5′-AACCGAGGGTGACGGGCGGTGTGTACC-3′) in the following conditions: 94°C/5 min—initial denaturation; 94°C/30 s, 55°C/45 s, and 72°C/1 min—40 cycles; 72°C/7 min—final extending (Wirtherle et al. 2007). After amplification products were purified using QIAquick PCR purification kit (Qiagen) and sequenced on Applied Biosystems ABI PRISM 3100-Avant DNA Sequencer.

In order to elucidate any homologies with previously deposited sequences in Gen Bank, we conducted a BLAST search (www.ncbi.nih.gov/BLAST/). The multiple alignment was done by the use of CLUSTAL W implemented to MEGA 4.0 package (Tamura et al. 2007) by the use of default parameters (gap opening penalty, 15; gap extension penalty, 6.66; both for pairwise and multiple alignment). The evolutionary distances were computed by Maximum Composite Likelihood Method with the complete deletion option. The neighbor joining (NJ) and maximum parsimony (MP) trees were constructed using MEGA 4.0 software. The tree was evaluated using the bootstrap test based on 2,000 resamplings. All obtained sequences are deposited in GenBank under accession numbers: JN088186–JN088190.

Results and discussion

As a result of the amplification of partial sequence of 18S rDNA, we obtained fragments of SSU from four isolates (1,126 bp—M. glareolus (RM), 1,140 bp—A. agrarius (WO), 1,116 bp—A. agrarius (WR), and 1,162 bp—M. glareolus (WR)). In relation to mitochondrial sequence, 354 bp product of 12S rDNA was obtained from one isolate (M. glareolus (RM)). Alignment of our four 18S rDNA sequences show 100% similarity in the overlapping regions with sequences of M. litteratus (DQ642999-DQ643002) from red foxes (Vulpes vulpes) from Slovakia, Czech Republic, and Spain (Literak et al. 2006). Comparison with shorter sequences of tetrathyridial isolates from sand lizard Lacerta agilis collected in Czech Republic (AY426257) also showed 100% similarity.

In the case of mitochondrial rDNA sequences, 99% similarity was obtained between isolates of adult tapeworms from red foxes (L49450) and tetrathyridia from dogs (EF567417) from Germany, and both included the species M. lineatus (Nickisch-Rosenegk et al. 1999; Wirtherle et al. 2007). Our isolate was 98% similar to sequences of M. litteratus from the Slovak Republic (JF268556–JF268580) and 68% similar to M. lineatus (JF268553–JF268555).

The final alignment of 18S rDNA sequences from 28 Mesocestoides spp. was 769 bp long, with 84 characters that were variable and 54 that were parsimony informative. The out-group for phylogenetic analysis is comprised two closely related species: Amurotaenia decidua (AF124474) and Nippotaenia chaenogobii (AF286987). Both NJ and MP tree-building methods showed similar results (Fig. 1a, b). Isolates were divided into two major groups, the first comprised all sequences from parasites obtained from red foxes in Europe (DQ642999–DQ643002), our isolates from rodents, and Mesocestoides spp. from L. agilis (AY426257). The second group was divided into five clades: (1) isolates of Mesocestoides corti, (2) Mesocestoides spp. from dogs and coyotes from the USA, (3) metacestodes form starling from the Czech Republic and dog in Italy, (4) isolates from A. agrarius from Bulgaria, and (5) Mesocestoides spp. from dogs from Germany.

Fig. 1
figure 1figure 1

Reconstruction of phylogenetic relationships within Mesocestoides spp., based on partial sequences of small subunit of rDNA, by the use of NJ (a) and MP (b) methods. The figure presents the bootstrap consensus trees with a cut-off value of 50%

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Phylogenetic analysis of Mesocestoides spp. based on mitochondrial 12S rDNA was performed with a 305-bp long alignment (107 characters were variable and 72 parsimony informative) comprising 33 sequences. The out-group for phylogenetic analysis is the closely related species Taenia taeniaeformis (EU219552.1). Trees obtained by the use of two tree-building methods have the same topology and we could distinguish two major groups, i.e., the first group, which include our isolate of tetrathyridium from M. glareous, M. litteratus (JF268556-JF268565), M. lineatus (L4951), and Mesocestoides leptothylacus (L49450); and the second group comprising isolates previously reported as clades A, B, and C (Crosbie et al. 2000; Padgett et al. 2005) and isolates of M. lineatus (JF268553–JF268555) (Fig. 2a, b).

Fig. 2
figure 2figure 2

Reconstruction of phylogenetic relationships within Mesocestoides spp., based on partial sequences mitochondrial 12S rDNA, by the use of NJ (a) and MP (b) methods. The figure presents the bootstrap consensus trees with a cut-off value of 50%

Full size image

Taxonomic identification of helminths is usually based on morphological features, but this approach is much more appropriate for adult parasites. The determining of species affinity of larval stages by morphometric criteria often presents difficulties. However, recent studies concerning the identification of larvae of Taenia spp., Trichinella spp., or members of Ascarididae show that implementation of molecular techniques contribute to our understanding of taxonomic relationships (Ishiwata et al. 2004; Malakauskas et al. 2007; Al-Sabi and Kapel 2011). Prior literature concerning the molecular analysis within the genus Mesocestoides is present. In 2000, Crossbie et al. conducted an analysis of sequence variation of Mesocestoides spp. parasitizing canids in western North America. The authors showed, based on 18S rDNA sequences, that all isolates of proglottids and tetratyridia collected from dogs and coyotes formed a monophyletic group and belonged to Mesocestoides genus. Additional analysis by the use of ITS2 marker revealed three distinct monophyletic groups (clades A, B, and C). Another study (Padgett et al. 2005) supported those clades and confirmed that Mesocestoides belonging to different clades are separate species. Molecular phylogeny indicated that clade B represents the species Mesocestoides vogae, whereas clades A and B, because of poor morphological data, could not be determined to the species level. Foronda et al. (2007) made a morphological and molecular identification of larval form of Mesocestoides spp. isolated from the peritoneal cavity of dogs and cats. As a result authors showed that isolates from Tenerife comprised the sequences which were distinct from those previously reported, and so it may be a different species than M. litteratus or representatives of clades A, B, and C.

The data concerning the taxonomic status of European isolates of Mesocestoides are scarce and mostly limited to three reports, mainly based on adult tapeworms from red foxes identified in the Czech and Slovak Republics (Hrckova et al. 2011; Literak et al. 2006; Literak et al. 2004) and one, canine peritoneal cestodosis, from the territory of Germany (Wirtherle et al. 2007). The results from studies of Literak et al. (2004; 2006) indicated that the European fox population is parasitized by M. litteraus, whereas Wirtherle et al. (2007), based on 12S rDNA sequence analysis, identified tetrathyridia from dogs as M. lineatus. The results presented in recent paper by Hrckova et al. (2011) indicate that the German isolates were misidentified and should be classified as M. litteratus. The only molecular evidence of tetrathyridia from rodents in Europe is provided by Literak et al. (2004), where authors, among others, analyzed the sequence of 18S rDNA isolated from A. agrarius from Bulgaria. Our isolate and those from Bulgaria are quite distinct (only 97% similarity) and it may constitute different species.

The results of our research on the larval stages from rodents, both living in a periphery of urban agglomeration as well as in an area of reserve protection, confirm the data of more frequently occurring of M. litteratus. Simultaneously, the data obtained in our studies confirmed incorrect determination of tapeworms described as M. lineatus and M. leptothylacus by German authors (Nickisch-Rosenegk et al. 1999; Wirtherle et al. 2007). The future studies should be conducted both on adult and larval stages using various molecular markers; these will allow for full identification of species and may explain ambiguities concerning the life cycle and life history of Mesocestoides.