[show abstract][hide abstract] ABSTRACT: Atractolytocestus tenuicollis (Li, 1964) Xi, Wang, Wu, Gao et Nie, 2009 is a monozoic, non-segmented tapeworm of the order Caryophyllidea, parasitizing exclusively common carp (Cyprinus carpio L.). In the current work, the first molec-ular data, in particular complete ribosomal internal transcribed spacer 2 (ITS2) and partial mitochondrial cytochrome c oxidase subunit I (cox1) on A. tenuicollis from Niushan Lake, Wuhan, China, are provided. In order to evaluate molecular interrela-tionships within Atractolytocestus, the data on A. tenuicollis were compared with relevant data on two other congeners, Atractolytocestus huronensis and Atractolytocestus sagittatus. Divergent intragenomic copies (ITS2 paralogues) were de-tected in the ITS2 ribosomal spacer of A. tenuicollis; the same phenomenon has previously been observed also in two other congeners. ITS2 structure of A. tenuicollis was very similar to that of A. huronensis from Slovakia, USA and UK; overall pairwise sequence identity was 91.7–95.2 %. On the other hand, values of sequence identity between A. tenuicollis and A. sagittatus were lower, 69.7–70.9 %. Cox1 sequence, analysed in five A. tenuicollis individuals, were 100 % identical and no intraspecific variation was observed. Comparison of A. tenuicollis cox1 with respective sequences of two other Atractolytocestus species showed that the mito-chondrial haplotype found in Chinese A. tenuicollis is struc-turally specific (haplotype 4; Ha4) and differs from all so far determined Atractolytocestus haplotypes (Ha1 and Ha2 for A. huronensis; Ha3 for A. sagittatus). Pairwise sequence identity between A. tenuicollis cox1 haplotype and remaining three haplotypes followed the same pattern as in ITS2. The nucle-otide and amino acide (aa) sequence comparison with A. huronensis Ha1 and Ha2 revealed higher sequence identity, 90.3–90.8 % (96.9 % in aa), while lower values were achieved between A. tenuicollis haplotype and Ha3 of Japanese A. sagittatus—75.2 % (81.9 % in aa). The phylogenetic analyses using cox1, ITS2 and combined cox1+ITS2 sequences re-vealed close genetic interrelationship between A. tenuicollis and A. huronensis. Independently of a type of analysis and DNA region used, the topology of obtained trees was always identical; A. tenuicollis formed separate clade with A. huronensis forming a closely related sister group.
Parasitology Research 10/2013; 112:3379-3388. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: The high-resolution melting (HRM) method, recently optimized as a reliable technique for population study of the European Fascioloides magna populations, was applied to determine an origin of F. magna individuals from Croatia. The structure and frequency of mitochondrial cytochrome c oxidase subunit I (439 bp; cox1) haplotypes of 200 Croatian flukes coming from 19 red deer (Cervus elaphus elaphus) livers were screened and compared with recently determined reference samples of F. magna from all European foci-Italy, Czech Republic, and Danube floodplain forests. While the reference haplotypes Ha1 and Ha2 were specific for flukes from the first European focus of fascioloidosis, the Natural Park La Mandria in Italy, the remaining three haplotypes (Ha3, Ha4, and Ha5) represented parasites from the second focus, Czech Republic. Besides, Ha3 and Ha4 were found also in the third, latest, and still expanding European focus, the Danube floodplain forests. The HRM screening of cox1 haplotypes of Croatian F. magna individuals resulted in classification of samples into the two mitochondrial haplogroups characterized by well-distinguished melting curves. They corresponded to Ha3 and Ha4 reference haplotypes that confirmed the Danube origin of F. magna from Croatia. The results support the theory that the Danube floodplain forests population of F. magna represents uniform genetic pool of the parasite. The spread of F. magna alongside the Danube River down to Croatia was possible due to suitable ecological conditions for definitive and intermediate hosts present in this unique biotope.
Parasitology Research 04/2013; · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: The sequence structure of the ribosomal internal transcribed spacer 2 (ITS2) was determined for six species of Khawia (Cestoda: Caryophyllidea), parasites of cyprinid fish in the Holarctic Region. Homologous intragenomic ITS2 structure was found in Khawia armeniaca, Khawia baltica, and Khawia rossittensis; whereas divergent intragenomic ITS2 copies were detected in Chinese, Japanese, and Slovak isolates of Khawia sinensis and in Khawia japonensis, both parasitic in common carp, and in Khawia saurogobii, recently described from Chinese lizard gudgeon in China. Despite distinct morphological differences between K. saurogobii and K. sinensis, both species display very high level of molecular homogeneity. Variation in number of short repetitive motifs [(GCCT)( n ) (GCCC)( n )], [(GTG)( n )], [(ATAC)( n )], [ACGTGT (TCGTGT)( n )], [(GT)( n )], [(GT)( n )], and [(ACCT)( n ) (GCCT)( n )] resulted in assortment of ITS2 sequences in four ITS2 variants in K. saurogobii from China, three in Chinese and Japanese isolates of K. sinensis, and five ITS2 variants in K. sinensis from Slovakia. In K. japonensis, the structure and arrangement of microsatellites was different from those of K. sinensis and K. saurogobii. The heterogeneity in the number of two microsatellite regions [(TG)( n ); (TTG)( n )] divided ITS2 clones into two variants-first ITS2 variant (472 bp) with (TG)(5) and (TTG)(6), and second variant with (TG)(7) and (TTG)(2) (465 bp). Sequence identity of K. saurogobii with all but one (K. sinensis) congeneric species ranged between 49.5 and 69.2 %, which corresponds to the interspecific differences. In contrast, sequence identity of K. saurogobii and K. sinensis (87.6-95.0 %) failed into the range of intraspecific variation determined for K. sinensis samples. This close genetic similarity indicates that recently described K. saurogobii may have undergone morphological divergence as a result of ongoing sympatric speciation by host switching.
Parasitology Research 07/2012; 111(4):1621-7. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chromosomal characteristics, i.e., number, size, morphology, and location of ribosomal DNA (rDNA) clusters were examined in
two medically important liver flukes, Fasciola hepatica and Fascioloides magna (Fasciolidae), using conventional Giemsa staining and fluorescent in situ hybridization (FISH) with ribosomal 18S rDNA probe.
A comparison of F. magna and F. hepatica karyotypes confirmed significant differences in all chromosomal features. Whilst the karyotype of F. hepatica comprised ten pairs of chromosomes (one metacentric and nine medium-sized subtelocentrics and submetacentrics; 2n = 20, n = 1 m + 5 sm + 4 st; TCL = 49.9μm), the complement of F. magna was composed of 11 pairs of medium-sized subtelocentrics and submeta-metacentrics (2n = 22, n = 9 st + 1 sm + 1 sm-m; TCL = 35.2μm). Noticeable differences were found mainly in length and morphology of first chromosome
pair. It was metacentric and 9.0μm long in F. hepatica while subtelocentric and 4.7μm long in F. magna. Although FISH with rDNA probe revealed a single cluster of ribosomal genes in both species, conspicuous interspecific differences
were displayed by chromosomal location of ribosomal loci (i.e., NORs). The signals were found on short arms of fifth homologous
pair in F. hepatica; however, they were detected in pericentromeric regions of the long arms of tenth pair in F. magna. The observed cytogenetic differences were interpreted in terms of karyotype evolution of fasciolid flukes; F. hepatica may be regarded phylogenetically younger than F. magna. The present paper provides a pilot study on molecular cytogenetics within a group of hermaphroditic digenetic flukes.
Parasitology Research 04/2012; 109(4):1021-1028. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Molecular comparative analysis of eggs of four liver and stomach flukes of cervids and domestic ruminants, Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi, was performed using a new methodological approach for eggshell disintegration. Eggs of all species were crushed mechanically
by the Teflon method (PTFE) without use of chemical reagents and an efficient disruption of eggshell was checked microscopically.
The egg suspension was then subjected to DNA isolation and PCR amplification using species-specific primers that annealed
to the internal transcribed spacer 2 (ITS2) region of ribosomal DNA. The size of PCR products of individual species corresponded
well to the size of amplicons obtained from adult flukes. The results provided evidence that the Teflon method does not destroy
the structure of egg DNA, thus making the procedure broadly applicable during coprological examinations. Molecular markers
introduced here are particularly important for blanket screening and differentiation of morphologically hardly distinguishable
F. hepatica, F. magna and P. cervi eggs.
Keywordsliver and stomach flukes-ruminants-eggs-ribosomal internal transcribed spacer 2-PTFE homogenizer
[show abstract][hide abstract] ABSTRACT: The species-specific ribosomal internal transcribed spacer 2 (ITS2) markers were designed for PCR-based molecular differentiation
of Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi, liver and stomach flukes of domestic and free living ruminants. Complete ITS2 sequences were obtained for D. dendriticum and P. cervi, for the later species, ITS2 structure was determined for the first time. Intraspecific variation within geographically distant
populations was found to be either very low (F. hepatica; D. dendriticum) or even absent (F. magna; P. cervi). ITS2 regions with the absence of intraspecific polymorphisms but with interspecific sequence heterogeneity were applied
for design of speciesspecific primers. The specificity of developed primers was tested on genomic DNA isolated from adult
individuals of studied fluke species. Application of the primers is of particular value for molecular differentiation of morphologically
hardly distinguishable F. hepatica, F. magna and P. cervi eggs after coprological examinations.
Keywordsliver and stomach flukes-ribosomal internal transcribed spacer-molecular markers-intraspecific variation
[show abstract][hide abstract] ABSTRACT: Caryophyllidean cestodes (Platyhelminthes) represent an unusual group of tapeworms lacking serially repeated body parts that potentially diverged from the common ancestor of the Eucestoda prior to the evolution of segmentation. Here we evaluate the utility of two nuclear and two mitochondrial molecular markers (ssrDNA and lsrDNA, nad3 and cox1) for use in circumscribing generic boundaries and estimating interrelationships in the group. We show that these commonly employed markers do not contain sufficient signal to infer well-supported phylogenetic estimates due to substitution saturation. Moreover, we detected multiple trnK+nad3+trnS+trnW+cox1 haplotypes within individuals, indicating a history of gene exchange between the mitochondrial and nuclear genomes. The presence of such nuclear paralogs (i.e. numts), to our knowledge described here in cestodes for the first time, together with the results of phylogenetic, saturation and split-decomposition analyses all suggest that finding informative markers for estimating caryophyllidean evolution is unusually problematic in comparison to other major lineages of tapeworms.
International journal for parasitology 02/2012; 42(3):259-67. · 3.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sequence structure of complete internal transcribed spacer 1 and 2 (ITS1 and ITS2) of the ribosomal DNA region and partial mitochondrial cytochrome c oxidase subunit I (cox1) gene sequences were studied in the monozoic tapeworm Atractolytocestus sagittatus (Kulakovskaya et Akhmerov, 1965) (Cestoda: Caryophyllidea), a parasite of common carp (Cyprinus carpio carpio L.). Intraindividual sequence diversity was observed in both ribosomal spacers. In ITS1, a total number of 19 recombinant clones yielded eight different sequence types (pairwise sequence identity, 99.7-100%) which, however, did not resemble the structure typical for divergent intragenomic ITS copies (paralogues). Polymorphism was displayed by several single nucleotide mutations present exclusively in single clones, but variation in the number of short repetitive motifs was not observed. In ITS2, a total of 21 recombinant clones yielded ten different sequence types (pairwise sequence identity, 97.5-100%). They were mostly characterized by a varying number of (TCGT)(n) repeats resulting in assortment of ITS2 sequences into two sequence variants, which reflected the structure specific for ITS paralogues. The third DNA region analysed, mitochondrial cox1 gene (669 bp) was detected to be 100% identical in all studied A. sagittatus individuals. Comparison of molecular data on A. sagittatus with those on Atractolytocestus huronensis Anthony, 1958, an invasive parasite of common carp, has shown that interspecific differences significantly exceeded intraspecific variation in both ribosomal spacers (81.4-82.5% in ITS1, 74.4-75.2% in ITS2) as well as in mitochondrial cox1, which confirms validity of both congeneric tapeworms parasitic in the same fish host.
Parasitology Research 10/2011; 110(5):1621-9. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: The giant liver fluke, Fascioloides magna, a liver parasite of free-living and domestic ruminants of Europe and North America, was analysed in order to determine the origin of European populations and to reveal the biogeography of this originally North American parasite on the European continent. The variable fragments of the mitochondrial cytochrome c oxidase subunit I (cox1; 384bp) and nicotinamide dehydrogenase subunit I (nad1; 405bp) were used. Phylogenetic trees and haplotype networks were constructed and the level of genetic structuring was evaluated using population genetic tools. In F. magna individuals originating from all European foci of infection (Italy, Czech Republic and Danube floodplain forests involving the territories of Slovakia, Hungary and Croatia) and from four of five major North American enzootic areas, 16 cox1 and 18 nad1 haplotypes were determined. The concatenated sequence set produced 22 distinct haplotypes. The European fluke populations were less diverse than those from North America in that they contained proportionately fewer haplotypes (eight), while a more substantial level of genetic diversity and a greater number of haplotypes (15) were recorded in North America. Only one haplotype was shared between the European (Italy) and North American (USA/Oregon and Canada/Alberta) flukes, supporting a western North American origin of the Italian F. magna population. Haplotypes found in Italy were distinct from those determined in the remaining European localities which indicates that introduction of F. magna to the European continent occurred more than once. In the Czech focus of infection, a south-eastern USA origin was revealed. Identical haplotypes, common to parasites from the Czech Republic and from an expanding focus in Danube floodplain forests, implies that the introduction of F. magna to the Danube region came from an already established Czech focus of infection.
International journal for parasitology 03/2011; 41(3-4):373-83. · 3.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: The invasive monozoic tapeworm Atractolytocestus huronensis, a specific parasite of common carp, was originally found and described in the North American continent. It has been introduced to Europe and reported in several countries in the last 15 years, as well. In the current study, tapeworms from one North American (USA) and five European localities (United Kingdom/UK, Slovakia, Hungary, Croatia, and Romania) were subjected to molecular analyses in order to determine the level of intrapopulation and intraspecific molecular variation and to assess interrelationships among American and European populations of the parasite. Partial sequences (672 bp) of the mitochondrial cytochrome c oxidase subunit I (cox1) revealed the presence of only two cox1 haplotypes, in accordance with the nonnative character of the populations. The first haplotype was common for all tapeworms from the Continental Europe (Slovakia, Hungary, Croatia, and Romania); no differences were determined either within or among respective A. huronensis populations. The second cox1 haplotype was characterized in all individuals from the USA and UK, indicating their close genetic relationship. Both haplotypes differed in three nucleotide positions (99.6% identity) which did not change the amino acid sequence. The cox1 data imply that introduction of the parasite to Europe was probably the result of two independent events directed to the UK and Continental Europe. The very close genetic relationship between British and American A. huronensis was reflected also by similar ribosomal internal transcribed spacer 2 (ITS2) sequence structure; considerable intragenomic ITS2 variability was detected in all individuals of both geographic populations. Divergent ITS2 copies were mostly induced by different numbers of short repetitive motifs within the sequences, allowing their assortment into two ITS2 variants.
Parasitology Research 01/2011; 109(1):125-131. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: The high-resolution melting (HRM) technique was successfully optimized as fast and effective method for population study of digenetic fluke, Fascioloides magna (Trematoda: Fasciolidae), originally North American liver parasite of free-living and domestic ruminants. Previously selected variable region (439 bp) of mitochondrial cytochrome c oxidase subunit I (cox1) of 249 fluke individuals from enzootic European and North American regions were sequenced and mutually compared. The sequence analysis of partial cox1 revealed presence of seven structurally different haplotypes. Based on the sequence structure and alignments of six of them (Ha1-Ha6), three internal probes were designed and applied in HRM-based haplotype determination of all F. magna specimens. HRM analysis, performed with three designed probes, resulted in classification of samples into the seven haplogroups, equally with their assortment according to the sequence analysis. The representative of the haplotype, which was not involved in probe design (Ha7), was characterized by a unique melting curve shape as well. This provided an evidence of optimally settled conditions in HRM assay and indicated a probability of successful discrimination of novel haplotypes in future population studies on F. magna. The successful optimization of HRM method stands for an opportunity of detection of genetically unknown North American variants of F. magna and promises its application as fast and cheap screening technique for phylogeography studies of the giant liver fluke on its original continent.
Parasitology Research 10/2010; 108(1):201-9. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Complete sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) and karyological characters of the monozoic (unsegmented) tapeworm Atractolytocestus huronensis Anthony, 1958 (Cestoda: Caryophyllidea) from Slovakia were analysed, revealing considerable intra-genomic variability and triploidy in all analysed specimens. Analysis of 20 sequences of each ITS1 and ITS2 spacer yielded eight and 10 different sequence types, respectively. In individual tapeworms, two to four ITS1 and three to four ITS2 sequence types were found. Divergent intra-genomic ITS copies were mostly induced by nucleotide substitutions and different numbers of short repetitive motifs within the sequence. In addition, triploidy was found to be a common feature of A. huronensis. The karyotype of Slovakian A. huronensis possesses three sets of chromosomes (3n=24, n=4m+3st+1minute chromosome), similar to the previously described triploidy in conspecific tapeworms from North America. Fluorescent in situ hybridisation (FISH) with a ssrDNA probe revealed two distinct rDNA clusters for each homologue of the triplet number 2. To date, A. huronensis is the only cestode species in which intra-individual ITS sequence variants were found in parallel with its triploid nature and multiple rDNA loci. Some of these molecular and genetic features were observed in several other species of basal or nearly basal tapeworms of the orders Caryophyllidea and Diphyllobothriidea, which indicates that the phenomena may be characteristic for evolutionarily lower tapeworms and deserve more attention in future studies.
International journal for parasitology 08/2009; 40(2):175-81. · 3.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Five horse farms located in eastern Slovakia were investigated for the presence of benzimidazole-resistant strongyles by faecal egg count reduction test and egg hatch assay. Coprocultures were prepared for each farm from faecal samples taken pre- and post-treatment and harvested larvae were molecularly examined with a Reverse Line Blot assay. Faecal egg count reduction values ranged from 0 to 52.5% and all farms were positive for benzimidazole-resistant cyathostomins. Seven benzimidazole-resistant cyathostomin species were molecularly identified on farms before and also after treatment. These data demonstrate that resistance to benzimidazoles is well established in cyathostomin populations from horse farms in the Slovak Republic and that the molecular assay was able to determine the species-specific distribution of resistant cyathostomins under field conditions.
[show abstract][hide abstract] ABSTRACT: Complete sequences of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna are presented. In particular, small subunit (18S) and internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene (rDNA), as well as cytochrome c oxidase subunit I (cox1) and nicotinamide dehydrogenase subunit I (nad1) of the mitochondrial DNA (mtDNA), were analyzed. The 18S and ITS sequences were compared with previously published sequences of the liver fluke Fasciola hepatica. Fixed interspecific genetic differences were determined that allow molecular differentiation of F. magna and F. hepatica using either the PCR-RFLP method or PCR amplification of species-specific DNA regions. Additionally, intraspecific sequence polymorphism of the complete cox1 and nad1 mitochondrial genes in geographically distinct F. magna populations was determined. Based on the sequence divergences, short (< 500 bp) variable regions suitable for broader biogeographical studies of giant liver fluke were designed.
Journal of Parasitology 03/2008; 94(1):58-67. · 1.32 Impact Factor