Sequence variability in three mitochondrial DNA regions of Spirometra erinaceieuropaei spargana of human and animal health significance

Sequence variability in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 3 (cox3), NADH dehydrogenase subunits 1 and 4 (nad1 and nad4) in Spirometra erinaceieuropaei spargana from different geographical regions in China was examined. A portion of each of the cox3 (pcox3), nad1 (pnad1) and nad4 genes (pnad4) were amplified separately from individual S. erinaceieuropaei spargana by polymerase chain reaction (PCR). Representative amplicons were subjected to sequencing in order to estimate sequence variability. The sequences of pcox3, pnad1 and pnad4 were 541, 607 and 847?bp in length, respectively. The A+T contents of the sequences were 68.39-68.76% (pcox3), 63.76-64.91% (pnad1) and 67.18-67.77% (pnad4), respectively, while the intra-specific sequence variations within each of the S. erinaceieuropaei spargana were 0-1.5% for pcox3, 0-2.8% for pnad1 and 0-2.7% for pnad4. Phylogenetic analysis using neighbour joining (NJ), maximum likelihood (ML) and maximum parsimony (MP) methods, indicated that all the spargana isolates in Hunan Province represented S. erinaceieuropaei. These findings demonstrated clearly the usefulness of the three mtDNA sequences for population genetics studies of S. erinaceieuropaei spargana of human and animal health significance.     

Sequence variability in three mitochondrial genes between the two pig nodule worms Oesophagostomum dentatum and O. quadrispinulatum

In this study, sequence variation in three mitochondrial DNA regions, namely cytochrome c oxidase subunit (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), between Oesophagostomum dentatum and O. quadrispinulatum isolated from pigs in different geographical origins in Mainland China was examined, and their phylogenetic relationships were reconstructed. A partial of the cox1 (pcox1), nad1, and nad4 genes (pnad1 and pnad4) were amplified separately from individual nodule worms by PCR and were subjected to direct sequencing in order to define sequence variations. While the intraspecific sequence variations within each of the two species were 0.3-5.2% for pcox1, 0-4.9% for pnad1, and 0-7.1% for pnad4, the interspecific sequence differences were significantly higher, being 10.7-13.4% for pcox1, 11-14.6% for pnad1, and 14.9-18% for pnad4, respectively. There were a number of nucleotide positions in the pcox1, pnad1, and pnad4 sequences with no apparent intraspecific variation but distinct interspecific differences among those samples of Oesophagostomum spp. examined, which may be used as genetic makers for the identification and differentiation of the Oesophagostomum spp. Phylogenetic analyses using three inference methods, namely Bayesian inference, maximum likelihood, and maximum parsimony based on the combined sequences of pcox1, pnad1, and pnad4 revealed that the O. dentatum and O. quadrispinulatum form monophyletic groups, respectively. These findings demonstrated clearly the usefulness of the three mitochondrial sequences for studying systematics, population genetic structures, and the molecular ecology of Oesophagostomum spp.     

Characterization of the complete mitochondrial genome sequence of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidae) from China

Sparganosis, caused by the plerocercoid larvae of members of the genus Spirometra, can cause significant public health problem and considerable economic losses. In the present study, the complete mitochondrial DNA (mtDNA) sequence of Spirometra erinaceieuropaei from China was determined, characterized and compared with that of S. erinaceieuropaei from Japan. The gene arrangement in the mt genome sequences of S. erinaceieuropaei from China and Japan is identical. The identity of the mt genomes was 99.1% between S. erinaceieuropaei from China and Japan, and the complete mtDNA sequence of S. erinaceieuropaei from China is slightly shorter (2 bp) than that from Japan. Phylogenetic analysis of S. erinaceieuropaei with other representative cestodes using two different computational algorithms [Bayesian inference (BI) and maximum likelihood (ML)] based on concatenated amino acid sequences of 12 protein-coding genes, revealed that S. erinaceieuropaei is closely related to Diphyllobothrium spp., supporting classification based on morphological features. The present study determined the complete mtDNA sequences of S. erinaceieuropaei from China that provides novel genetic markers for studying the population genetics and molecular epidemiology of S. erinaceieuropaei in humans and animals.     

Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids

We report the development of a colourimetric PCR/dot blot assay targeting the mitochondrial gene NADH dehydrogenase subunit 1 (nad1) for differential diagnosis of taeniid eggs. Partial sequences of the cestode nad1 gene were aligned and new primers were designed based on conserved regions. Species-specific oligonucleotide probes (S-SONP) for canine taeniid cestodes were then designed manually based on the variable region between the conserved primers. Specifically, S-SONP were designed for the Taenia crassiceps, T. hydatigena, T. multiceps, T. ovis, T. taeniaeformis, Echinococcus granulosus (genotype 1), E. multilocularis and E. vogeli. Each probe showed high specificity as no cross-hybridisation with any amplified nad1 fragment was observed. We evaluated the assay using 49 taeniid egg-positive samples collected from dogs in Zambia. DNA from 5 to 10 eggs was extracted in each sample. Using the PCR/dot blot assay, the probes successfully detected PCR products from T. hydatigena in 42 samples, T. multiceps in 3 samples, and both species (mixed infection) in the remaining 4 samples. The results indicate that the PCR/dot blot assay is a reliable alternative for differential diagnosis of taeniid eggs in faecal samples.     

A phylogeny of members of the family Taeniidae based on the mitochondrial cox1 and nad1 gene data

The cestode family Taeniidae consists of 2 genera, Taenia and Echinococcus, which both have been the focus of intensive taxonomic and epidemiological studies because of their zoonotic importance. However, a comprehensive molecular phylogeny of this family has yet to be reconstructed. In this study, 54 isolates representing 9 Taenia species were characterized using DNA sequences in the mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes. Phylogenetic relationships within the family Taeniidae were inferred by combining cox1 and nad1 sequence data of the present and previous studies. In the phylogenetic analysis, the genus Echinococcus was shown to be monophyletic, but Taenia proved to be paraphyletic due to the position of T. mustelae as a probable sister taxon of Echinococcus. This indicates that T. mustelae should form a genus of its own. Taenia ovis krabbei was placed distant from T. ovis ovis, as a sister taxon of T. multiceps, supporting its recognition as a distinct species, T. krabbei. High intraspecific sequence variation within both T. polyacantha and T. taeniaeformis suggests the existence of cryptic sister species.     

猪鞭虫和斯氏鞭虫线粒体pnad4和pcox1基因的扩增与序列分析

以采自广东不同地区的猪鞭虫(Trichuris suis)和斯氏鞭虫(Trichuris skrjabini)为研究对象,扩增线粒体基因组的pnad4和pcox1基因,将扩增产物纯化后克隆至pMD18-T载体,对阳性菌落进行测序和序列分析。结果表明,来源于不同地区的鞭虫上述两个基因种内相对保守,差异性不大,但种间差异较大。猪鞭虫和斯氏鞭虫的pnad4长度均为468 bp,相互间有162个种间遗传标记位点;猪鞭虫和斯氏鞭虫的pcox1长度分别为600 bp与438 bp,相互间有156个种间遗传标记位点。两种鞭虫之间的pnad4基因差异率为45.1%-45.9%,pcox1基因差异率为44.1%-45.4%。上述线粒体两个基因均可作为区分两种线虫的种间遗传标记。     

Complete sequence of the mitochondrial genome of Taenia saginata: comparison with T. solium and T. asiatica

The complete sequence of the Taenia saginata mitochondrial genome was determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The mitochondrial genome was 13,670 bp long, contained 12 protein-coding genes, two ribosomal RNAs (rRNAs, a small and a large subunit), and 22 transfer RNAs (tRNAs). It did not encode the atp8 gene. Overlapping regions were found between nad4L and nad4, nad1 and trnN, and cox1 and trnT. The ATG initiation codon was used for 10 protein-coding genes, and the GTG initiation codon was used for the remaining 2 genes (nad4 and atp6). The size of the protein-coding genes of the three human Taenia tapeworms did not vary, except for Taenia solium nad1 (891 aa) and nad4 (1212 aa) and Taenia asiatica cox2 (576 aa). The tRNA genes were 57-75 bp long, and the predicted secondary structures of 18 of these genes had typical clover-leaf shapes with paired dihydrouridine (DHU) arms. The genes in all human Taenia tapeworms for the two mitochondrial rRNA subunits rrnL and rrnS are separated by trnC. The putative T. saginata rrnL and rrnS are 972 and 732 bp long, respectively. The non-coding regions of the mt genome of T. saginata consisted of 2 regions: a short non-coding region (SNR, 66 nucleotides) and a long non-coding region (LNR, 159 nucleotides). The overall sequence difference in the full mitochondrial genome between T. saginata and T. asiatica was 4.6%, while T. solium differed by 11%. In conclusion, the complete sequence of the T. saginata mitochondrial genome will serve as a resource for comparative mitochondrial genomics and systematic studies of the parasitic cestodes.     

Sequence analysis of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna (Trematoda: Fasciolidae): intraspecific variation and differentiation from Fasciola hepatica

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.     

Intraspecific variation of Spirometra erinaceieuropaei and phylogenetic relationship between Spirometra and Diphyllobothrium inferred from mitochondrial CO1 gene sequences

Spirometra erinaceieuropaei is a diphyllobothriid cestode whose adult stage occurs mainly in cat-like carnivores, but occasionally in canids and humans. Although it is generally accepted that the distribution of S. erinaceieuropaei is cosmopolitan, it is controversial as to whether all of S. erinaceieuropaei reported are the same species. This study determined partial sequences of the CO1 gene from several isolates in Asian countries and compared them to sequence data from the GenBank/EMBL/DDBJ nucleotide sequence database. Then intraspecific variation of S. erinaceieuropaei and its phylogenetic relationship with Diphyllobothrium were evaluated. The level of nucleotide variation in the CO1 gene sequences within S. erinaceieuropaei was less than 2.6%. Although it was a little larger than that within each species of Diphyllobothrium (0.1-1.0%), it was much smaller than the interspecific variation within the genus Diphyllobothrium (6.2-14%). These facts indicate that all isolates of S. erinaceieuropaei used in this study, which were collected from Asia, Australia and New Zealand, belong to the same species. Based on CO1 gene sequences, genus Spirometra is clearly separate from the genus Diphyllobothrium. It seems that the genus Spirometra is not a synonym of the genus Diphyllobothrium. The phylogenetic relationship between S. erinaceieuropaei and Sparganum proliferum inferred from the CO1 gene clearly confirm the previous opinion that S. proliferum is a distinct species from S. erinaceieuropaei.     

Helminthologic survey of the wolf (Canis lupus) in Estonia, with an emphasis on Echinococcus granulosus

Carcasses of 26 wolves were collected during the 2000/2001 and 2003/2004 hunting seasons and examined for helminths. Thirteen helminth species were recorded: one trematode (Alaria alata), seven cestodes (Diphyllobothrium latum, Mesocestoides lineatus, Taenia hydatigena, Taenia multiceps, Taenia ovis, Taenia pisiformis, and Echinococcus granulosus), and five nematode species (Uncinaria stenocephala, Toxascaris leonina, Toxocara canis, Trichinella nativa, and Trichinella britovi). The most common species were A. alata and U. stenocephala. Mature Echinococcus granulosus was found and described for the first time in Estonia, and its identity verified using PCR-RFLP analysis. Sequencing a fragment of the mitochondrial DNA NADH dehydrogenase 1 (mtND1) gene showed that the E. granulosus strain from Estonia was identical to strain G10, recently characterized in reindeer and moose in Finland.     

Parasites present in foxes (Vulpes vulpes) of the province of Forli

In the period March 1983 - March 1984 a parasitological survey was carried out on 103 foxes killed in the Forli province (Italy). The parasites identified were: Toxocara canis (45.6%); Uncinaria stenocephala (14.6%); Mesocestoides lineatus (9.7%); Dipylidium caninum (2.9%); Taenia crassiceps (2.9%); Trichuris vulpis (2.9%). Out of all the cestodes found in 7 animals the authors identified only genus Taenia since their preservation conditions were not optimal. Other 13 subjects contained many cestodes similar to T. hydatigena, but considerably shorter in the mean length (20 cm vs 200). In a fecal sample the authors found a species of coccidium whose features do not correspond to any of those described in foxes, therefore it was called Eimeria sp. Finally, the autopsies' results were compared with coprological ones in order to asses their reliability.     

Helminths in the wolf, Canis lupus, from north-western Spain

Fifteen helminth species were collected from 47 wolves (Canis lupus ) which were surveyed from 1993 to 1999 in northwestern Spain. These included the trematode Alaria alata (2.1%); the cestodes Taenia hydatigena (44.7%), T. multiceps (29.8%), T. serialis (2.1%), Dipylidium caninum (6.4%) and Mesocestoides sp. aff. litteratus (4.2%); and the nematodes Pearsonema plica (7.4%), Trichuris vulpis (10.6%), Trichinella britovi (12.8%), Ancylostoma caninum (8.5%), Uncinaria stenocephala (51.1%), Toxocara canis (6.4%) Toxascaris leonina (4.2%), Angiostrongylus vasorum (2.1%) and Dirofilaria immitis (2.1%). Only two wolves were not infected. A single infection occurred in 28.9% of the cases, but the commonest infracommunity (31.1%) involved three species. The helminths Alaria alata, Taenia hydatigena, Mesocestoides sp. aff. litteratus, P. plica, Trichuris vulpis, and Ancylostoma caninum parasitizing C. lupus are reported for the first time in Spain. Taenia serialis and D. immitis are reported for the first time in wolves in Europe. Angiostrongylus vasorum represents a new host record for wolves. The helminth fauna of Spanish wolves is compared with that of other European wolf populations. Some epidemiological considerations of the helminth fauna of wolves in Spain and the health risk to humans are also discussed.     

Mitochondrial DNA divergence in populations of the tapeworm Diphyllobothrium nihonkaiense and its phylogenetic relationship with Diphyllobothrium klebanovskii

Diphyllobothrium nihonkaiense [Y. Yamane, H. Kamo, G. Bylund, J.P. Wilkgren. Diphyllobothrium nihonkaiense sp. nov (Cestoda: Diphyllobothriidae)- revised identification of Japanese broad tapeworm. Shimane J Med Sci 1986;10:29-48.] and Diphyllobothrium klebanovskii [I.V. Muratov, P.S. Posokhov. Causative agent of human diphyllobothriasis - Diphyllobothrium klebanovskii sp. n. Parazitologiia. 1988;22:165-170.] are two major species of human diphyllobothriasis in Japan and Far East Russia, respectively, but their taxonomical relationship remains unclear. In this study, we analysed the DNA sequences of 16 clinical isolates of D. nihonkaiense from Japanese people, 3 isolates of D. klebanovskii from a bear in Kamchatka, and 4 clinical isolates of D. klebanovskii from native Udygeyci people in Russia, as well as 4 plerocercoids from Oncorhynchus spp. 18S rDNA and internal transcribed spacer 1 (ITS1) sequences from D. nihonkaiense and D. klebanovskii showed a high level of similarity, indicating synonymy of the two species. Analyses of mitochondrial DNA (mtDNA) sequence polymorphisms in the cox1 and nad3 genes of D. nihonkaiense (D. klebanovskii) revealed two deeply divergent lineages, A and B, with genetic distances (Kimura-2 parameter) of 0.018-0.022. Furthermore, the distinct monophyletic groupings of cox1 haplotypes corresponded to the distinct monophyletic groupings of nad3 haplotypes. The two lineages were neither distinguished by morphological features nor defined by the localities of the samples. These results suggest that the two morphologically cryptic lineages have diverged and coexisted over a long period of time.     

Molecular evidence that Langeronia macrocirra and Langeronia cf. parva (Trematoda: Pleurogenidae) parasites of anurans from Mexico are conspecific

The genus Langeronia parasitizing the intestine of several species of anurans is distributed from North to Central America. We identified Langeronia macrocirra and Langeronia cf. parva from the same host and localities, and present here new data not applicable about their tegumental surface by scanning electron microscopy. We compared sequences of the rDNA ITS2 region and mtDNA cox1 gene for the two morphotypes. ITS2 exhibited a high degree of conservation. Phylogenetic reconstruction using cox1 revealed three clades (I, II, and III), which did not correspond to a previous identification or host. Little divergence was found within clades: sequences were identical in clade I, whereas clade II had 0.27% and clade III had 1.08%. Inter-clade divergence reached 8.69% (I vs. III). This pattern of genetic divergence indicated that both taxa probably belong to the same species, so we posit that the morphological changes could be correlated with development. Increasing sample size and geographical coverage will contribute to the taxonomy of the genus based on morphological and molecular evidence, and will open tracks toward the use of DNA barcodes to the genus in Mexico.     

Intestinal cestodes of stray dogs in Jordan

Five species of cestodes namely Echinococcus granulosus, Taenia hydatigena, Taenia pisiformis, Taenia ovis and Dipylidium caninum were recovered post mortem from 120 out of 173 stray dogs collected from the 5 governorates of Jordan during the period June 1979 to November 1980. Twenty-five of the examined dogs (14%) were found to be infected with E. granulosus, 79 (46%) with T. hydatigena, 14 (8%) with T. pisiformis and 5 (3%) with T. ovis. Dipylidium caninum was encountered in 33 (19%) of the examined dogs and infection with this parasite was significantly higher in males than in females. The parasites, except for D. caninum which was encountered in the ileum, were almost exclusively recovered from the duodenum and the jejunum. Single, double and triple infections with those cestodes were recorded.     

Molecular identification of a causative parasite species using formalin-fixed paraffin embedded (FFPE) tissues of a complicated human pulmonary sparganosis case without decisive clinical diagnosis

PCR-based molecular diagnosis was made for the identification of causative agents of the clinically suspected pulmonary proliferative sparganosis case found in Thailand using formalin-fixed paraffin-embedded (FFPE) biopsy specimens. As a reference, FFPE biopsy specimen from a typical cutaneous sparganosis case was examined together. DNA samples were extracted from tissues and two partial fragments of cytochrome c oxidase subunit 1 (cox1) gene were amplified for the detection of Spirometra DNA. Two cox1 fragments were amplified successfully for both specimens. After alignment of nucleotide sequences of the PCR-amplicons, the causative agents of both cases were identified as Spirometra erinaceieuropaei.     

Extensive variation in nuclear mitochondrial DNA content between the genomes of Phytophthora sojae and Phytophthora ramorum

Fragments of mitochondrial DNA (mtDNA) transferred to the nuclear genome are called nuclear mitochondrial DNAs (NUMTs). We report here a comparison of NUMT content between genomes from two species of the same genus. Analysis of the genomes of Phytophthora sojae and P. ramorum revealed large differences in the NUMT content of the two genomes: 16.27 x 10(-3) and 2.28 x 10(-3)% of each genome, respectively. Substantial differences also exist between the two species in the sizes of the NUMTs found in each genome, with ranges of 20 to 405 bp for P. sojae and 19 to 137 bp for P. ramorum. Furthermore, in P. sojae, fragments from the mitochondrial genes rns, rnl, coxl, and nad (various subunits) are found most frequently, whereas P. ramorum NUMTs most often originate from the cox3, rpsl4, nad4, and nad5 genes. The large differences in the presumptive mtDNA insertions suggest that the insertions occurred subsequent to the divergence of the two species, and this is supported by sequence comparisons among the NUMTs and the mtDNA sequences of the two species. P. sojae mtDNA sequences inserted in the nuclear genome appear to have been altered as a result of insertions, deletions, inversions, and translocations and provide insights into active mechanisms of sequence divergence in this plant pathogen. No clear examples were found of NUMTs forming functional nuclear genes or of NUMTs inserted into exons or introns of any nuclear gene.     

Nuclear ribosomal DNA monophyly versus mitochondrial DNA polyphyly in two closely related mite species: the influence of life history and molecular drive

In two very closely related but reproductively isolated mite species, Tetranychus urticae and T. turkestani, we found nucleotide diversity to be extensive for mitochondrial DNA (mtDNA) cytochrome oxidase 1 (COI) (3-4%) but extremely reduced for nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS2) (less than 0.5%). By contrast, ITS2 was shown to evolve much faster than COI between species of this genus. Furthermore, we found that these two species are polyphyletic for mtDNA but monophyletic for rDNA. Thus it appears that despite its biparental transmission and multiplicity of copies in the genome, nuclear rDNA has a smaller effective population size than mtDNA in these species. The conjunction of efficient concerted evolution and/or gene conversion in the rDNA cluster, the haplodiploidy of these species and their female-biased sex ratio could account for this apparent contradiction.     

Mitochondrial DNA sequence variation among geographical isolates of Opisthorchis viverrini in Thailand and Lao PDR, and phylogenetic relationships with other trematodes

The present study compared the genetic variation among 14 different geographical isolates of Opisthorchis viverrini sensu lato from Thailand and Lao PDR using sequence data for 2 mitochondrial DNA genes, the subunit 1 of NADH dehydrogenase gene (nad1) and cytochrome c oxidase gene (cox1). Four different nad1 haplotypes were detected among isolates, all of which were identical at the amino acid sequence level. Nucleotide sequence variation among 14 isolates ranged from 0 to 0.3% for nad1. Two different cox1 haplotypes were detected among isolates. These two haplotypes differed at 2 nucleotide positions, one of which resulted in a change in the amino acid sequence. Nucleotide sequence variation among isolates for cox1 ranged from 0 to 0.5%. Comparison of cox1 sequences of O. viverrini to those of other trematodes revealed nucleotide differences of 13-31%. A phylogenetic analysis of the cox1 sequence data revealed strong statistical support for a clade containing O. viverrini and 2 other species of opisthorchid trematodes; O. felineus and Clonorchis sinsensis.