Phylogenetic identification of Sparganum proliferum as a pseudophyllidean cestode by the sequence analyses on mitochondrial COI and nuclear sdhB genes.

Sparganum proliferum is a larval cestode for which the adult stage is unknown. It is characterized by the continuous branching and budding when parasitized to humans, and causes fatal human sparganosis. However, the biological features of S. proliferum, including its taxonomic status, still remain obscure. Our previous investigation suggested that S. proliferum might be phylogenetically distinct from Spirometra erinaceieuropaei, by the analysis on mitochondrial NADH dehydrogenase subunit 3 (ND3) gene. However, mitochondrial DNA sequence in Platyhelminth is known to have heteroplasmy within a species. Therefore, in the present study, we have investigated the complete nucleotide sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene and the partial nucleotide sequences of nuclear coded succinate dehydrogenase iron-sulfur protein subunit gene (sdhB). The results clearly demonstrated that S. proliferum is a distinct species from S. erinaceieuropaei, and that S. proliferum belongs to the order Pseudophyllidea.     

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.     

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.     

Use of nuclear and mitochondrial DNA PCR and sequencing for molecular identification of Diphyllobothrium isolates potentially infective for humans

Tapeworms of the genus Diphyllobothrium (Cobold, 1858) are widely distributed all around the world and some of them are agents of human diphyllobothriasis. Approximately 50 species have been described within the Diphyllobothrium genus but only 13 are human pathogens. Species identification by using morphological criteria is very difficult. We determined the value of 18S ribosomal RNA gene, internal transcribed spacer (ITS) and cytochrome c oxidase subunit 1 gene (COI) sequences to differentiate between Diphyllobothrium isolates. Sequences from 18 isolates (larvae or adults) of D. latum, D. nihonkaiense, D. ditremum, D. dentriticum and D. stemmacephalum species were obtained. COI region sequences analysis was clearly more discriminative than those of the ITS1 and 18S rRNA and was a useful tool for identifying specimens.     

Spirometra species from Asia: Genetic diversity and taxonomic challenges

Despite considerable controversy concerning the taxonomy of species within the genus Spirometra, human sparganosis and spirometrosis mainly in Asia and Europe has long been confidently ascribed to Spirometra erinaceieuropaei. Recently, the mitochondrial genomes of purported “S. erinaceieuropaei”, “Spirometra decipiens” and “Spirometra ranarum” from Asia have been determined. However, it has been pointed out that the morphological criteria used for identifying these species are unsuitable and thus these identifications are questionable. In the present study, therefore, Spirometra samples from Asia were re-examined based on mitochondrial cytochrome c oxidase subunit 1 gene sequences and the identification of these species was discussed. Haplotype network and phylogenetic analyses revealed that: i) two distinct Spirometra species, Type I and Type II, are present in Asia and neither of which is close to likely European “S. erinaceieuropaei”; ii) Type I is genetically diverse and widely distributed, however Type II is known so far from Japan and Korea; iii) “S. decipiens” and “S. ranarum” reported from Asia are conspecific with Type I; iv) Type I is probably conspecific with Spirometra mansoni, and Type II may represent an undescribed species.     

Sequence variability in two mitochondrial DNA regions and internal transcribed spacer among three cestodes infecting animals and humans from China

Sequence variability in two mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 4 (nad4), and internal transcribed spacer (ITS) of rDNA among and within three cestodes, Spirometra erinaceieuropaei, Taenia multiceps and Taenia hydatigena, from different geographical origins in China was examined. A portion of the cox1 (pcox1), nad4 genes (pnad4) and the ITS (ITS1+5.8S rDNA+ITS2) were amplified separately from individual cestodes by polymerase chain reaction (PCR). Representative amplicons were subjected to sequencing in order to estimate sequence variability. While the intra-specific sequence variations within each of the tapeworm species were 0-0.7% for pcox1, 0-1.7% for pnad4 and 0.1-3.6% for ITS, the inter-specific sequence differences were significantly higher, being 12.1-17.6%, 18.7-26.2% and 31-75.5% for pcox1, pnad4 and ITS, respectively. Phylogenetic analyses based on the pcox1 sequence data revealed that T. multiceps and T. hydatigena were more closely related to the other members of the Taenia genus, and S. erinaceieuropaei was more closely related to the other members of the Spirometra genus. These findings demonstrated clearly the usefulness of mtDNA and rDNA sequences for population genetic studies of these cestodes of socio-economic importance.     

基于线粒体CO 1基因序列的DNA条形码在鲤科鲌属鱼类物种鉴定中的应用

本研究探讨了线粒体CO1基因作为DNA条形码对鲌属鱼类进行物种鉴定的可行性。研究中获得了鲌属4种鱼类共32个个体长度为816bp的CO1基因序列。利用MEGA软件计算鲌属鱼类种间及种内遗传距离,利用邻接法、最大简约法、最大似然法和Bayesian方法分别构建分子系统树。结果显示,鲌属鱼类的种间遗传距离显著大于种内遗传距离。在系统树中,鲌属鱼类每一物种的个体分别形成各自独立的分支。基于CO1基因的DNA条形码在识别鲌属鱼类物种方面和传统形态学基本一致,而且该基因可以探讨鲌属鱼类种间的系统发育关系。本研究表明以CO1基因作为鲌属鱼类DNA条形码进行物种鉴定具有一定的可行性。     

Survey of Spirometra erinaceieuropaei in frogs in Taiwan and its experimental infection in cats

Eighteen of 56 (32.1%) wild Rana limnocharis from central and south Taiwan were found to contain plerocercoids of Spirometra erinaceieuropaei. This is the first report of S. erinaceieuropaei infections in frogs in Taiwan, with the plerocercoids being recovered from the thigh and back muscles or under the skin. Other species of frogs examined, including nine wild R. latouchii, one wild Buergeria robustus and 110 cultured R. rugulosa were free of infection. The plerocercoids were orally inoculated into four cats; three of which were each given a single plerocercoid and one a dose of three plerocercoids. Daily faecal examination showed that two cats started shedding eggs of S. erinaceieuropaei on day 8 postinfection (PI) and the other two on day 10 PI. The highest eggs per gram and eggs per day for a single worm was found to be 428,000 and 14,416,000 respectively. Only the cat inoculated with three plerocercoids shed proglottids in its faeces during the 2 month observation period.     

Molecular taxonomic position of the elephant schistosome, Bivitellobilharzia nairi, newly discovered in Sri Lanka

Bivitellobilharzia nairi (Mudaliar and Ramanujachar, 1945) Dutt and Srivastava, 1955 was first recorded in India. A number of adult worm specimens of this schistosome species were recovered from a domestic elephant, which died in 1999 in Sri Lanka. This is the first report of this schistosome from Sri Lanka. In the present study, in order to clarify the phylogenetic relationship with other species of schistosomes, sequences from the second internal transcribed spacer (ITS2) of the ribosomal gene repeat, part of the 28S ribosomal RNA gene (28S), and part of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene from B. nairi were analyzed. Two intraspecific variations were seen within 13 individuals in the ITS2 region. In the CO1 region of the mitochondrial DNA, there were four haplotypes in the nucleotide sequences and two haplotypes in the amino acid sequences. Phylogenetic analysis using the nuclear DNA showed that B. nairi was basal to all of species of the genus Schistosoma. The 28S tree also showed that the mammalian lineage was monophyletic. However, phylogenetic analysis using the mitochondrial DNA showed that B. nairi was nested within the genus Schistosoma. The taxonomical position for this species as well as the contradiction between the results from the nuclear and mitochondrial genes were discussed.     

Affinities between Asian non-human Schistosoma species,the S. indicum group,and the African human schistosomes

Schistosoma species have traditionally been arranged in groups based on egg morphology, geographical origins, and the genus or family of snail intermediate host. One of these groups is the 'S. indicum group' comprising species from Asia that use pulmonate snails as intermediate hosts. DNA sequences were obtained from the four members of this group (S. indicum, S. spindale, S. nasale and S. incognitum) to provide information concerning their phylogenetic relationships with other Asian and African species and species groups. The sequences came from the second internal transcribed spacer (ITS2) of the ribosomal gene repeat, part of the 28S ribosomal RNA gene (28S), and part of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. Tree analyses using both distance and parsimony methods showed the S. indicum group not to be monophyletic. Schistosoma indicum, S. spindale and S. nasale were clustered among African schistosomes, while S. incognitum was placed as sister to the African species (using ITS2 and 28S nucleotide sequences and CO1 amino acid sequences), or as sister to all other species of Schistosoma (CO1 nucleotide sequences). Based on the present molecular data, a scenario for the evolution of the S. indicum group is discussed.     

New Paracoccidioides brasiliensis isolate reveals unexpected genomic variability in this human pathogen

By means of genealogical concordance phylogenetic species recognition (GCPSR), we have investigated coding and non-coding regions from various genes and the ITS sequences of 7 new and 14 known isolates of Paracoccidioides brasiliensis. Such isolates grouped within the three phylogenetic groups recently reported in the genus Paracoccidioides, with one single exception, i.e., Pb01, a strain that has been the subject of intense molecular studies for many years. This isolate clearly separates from all other Paracoccidioides isolates in phylogenetic analyses and greatly increases the genomic variation known in this genus.     

Nucleotide sequence diversity of rDNA internal transcribed spacers extracted from conidia and cleistothecia of several powdery mildew fungi

An improved protocol, including DNA extraction with Chelex, two amplifications with a nested primer set, and DNA purification by electrophoresis, made it possible to analyze nuclear rDNA sequences of powdery mildew fungi using at most several hundred conidia or 20 cleistothecia. Nucleotide sequence diversity of the nuclear rDNA region containing the two internal transcribed spacers (ITS1 and ITS2) and 5.8S rRNA gene derived from conidia and cleistothecia was investigated for four kinds of powdery mildew fungi including two isolates of the same species. The results showed that the nucleotide sequences of the nuclear rDNA region were highly conserved between the teleomorph and the anamorph. Thus, the nucleotide sequence data obtained from either developmental stage can be used for phylogenetic studies of powdery mildew fungi. The nucleotide sequences of the 5.8S rRNA genes of the four species were highly conserved, but those of their ITS regions were variable. This suggests that the nuclear rDNA region is not suitable for phylogenetic studies of distantly related powdery mildew fungi, because too much sequence diversity exists, within the ITS, and too little phylogenetic information is contained within the 5.8S rRNA gene. However, the ITS region will be useful for phylogenetic comparison of closely related species or intraspecies. Contribution No. 132 from the Laboratory of Plant Pathology, Mie University.     

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.     

dnaJ and gyrB gene sequence relationship among species and strains of genus Streptococcus

The dnaJ and gyrB nucleotide sequences were determined for members of the genus Streptococcus. The average similarity between the species tested was 76.4% (69.7-100%) for dnaJ and 75.9 (70.1-98.7%) for gyrB. These data indicated that the dnaJ and gyrB genes are more divergent and more discriminatory than the 16S rDNA gene. Furthermore, the variation in the dnaJ nucleotide sequences among the mitis group was greater than that of the gyrB nucleotide sequences, especially between Streptococcus pneumoniae and Streptococcus mitis. Subsequently, the high discrimination power of dnaJ within the mitis group was confirmed. Thus, we conclude that the dnaJ and gyrB genes are efficient alternative targets for the classification of the genus Streptococcus, and that dnaJ is suitable for phylogenetic analysis of closely related Streptococcus strains.     

Phylogeny of Bipolaris inferred from nucleotide sequences of Brn1, a reductase gene involved in melanin biosynthesis

The Brn1 reductase melanin biosynthesis gene in the fungal genus Bipolaris was sequenced in 74 strains of 22 species. The Brn1 region was highly conserved among the species examined at the nucleotide and the amino acid levels. To elucidate the phylogenetic relationships among Bipolaris species, trees were inferred from nucleotide sequences of this region. Species in these trees formed exclusive clusters clearly separated from one another, except for B. panici-miliacei and B. setariae, and B. victoriae and B. zeicola. When unidentified strains were added to this tree, they fell within known species or formed independent clusters. These data indicated that the Brn1 gene region was suitable for species-level systematics within the genus. The results also suggest that Bipolaris consists of two or more clades that may reflect teleomorphic connections.     

Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis

We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.     

The marine pathogenic genotype of Spironucleus barkhanus from farmed salmonids redescribed as Spironucleus salmonicida n. sp

There are two genotypes of the diplomonad Spironucleus barkhanus. Based on sequence data from the small subunit ribosomal RNA gene the conspecificity of these two genotypes has been questioned. Therefore, we have sampled Spironucleus from 27 fish, representing 14 populations, five species, and four genera. Partial nucleotide sequences from the three genes; small subunit ribosomal DNA, glutamate dehydrogenase 1 and alpha-tubulin were compared. The pathogenic isolates of S. barkhanus, which causes systemic spironucleosis in Atlantic salmon, Chinook salmon, and Arctic charr, all farmed in sea water, were genetically very different from the commensal isolate found in wild freshwater populations of Arctic charr and grayling. The genetic distances between the genotypes were of the same magnitude as those separating species of Giardia. Based on these genetic and ecological data, the pathogenic genotype from farmed salmonids is described as a new species, Spironucleus salmonicida n. sp. Scanning and transmission electron microscopy showed no specific morphological or ultrastructural features distinguishing S. salmonicida n. sp. from S. barkhanus. The present study clearly demonstrates the value of applying genetics in identification of Spironucleus species. Phylogenetic analyses that included the isolates of S. salmonicida n. sp. did not change the phylogenetic relationship within the genus Spironucleus.     

New evolutionary lineages, unexpected diversity, and host specificity in the parabasalid genus Tetratrichomonas

We studied morphological and molecular polymorphism of 53 Tetratrichomonas isolates obtained from amphibian, reptilian, mammalian hosts, and from a slug with the aid of protargol staining and analyses of ITS1-5.8S rRNA-ITS2, SSU rRNA, and alpha-tubulin gene sequences. The phylogenetic tree based on the concatenate of all sequences showed the monophyly of the genus Tetratrichomonas with respect to the genus Trichomonas. Our data suggest that two parabasalid genera, Pentatrichomonoides and Trichomonoides, may belong to the genus Tetratrichomonas. Tetratrichomonas isolates were divided into 16 robust host-specific and monophyletic groups that probably represent separate, mostly new, species. As only five Tetratrichomonas species were described from the examined host taxa so far, our study uncovered considerable species diversity within the genus. The wide host range, high level of species-specific host specificity, and newly revealed biodiversity make the genus Tetratrichomonas a valuable model for studying evolution of parasites.     

Mitochondrial 16S rRNA sequence diversity of hominoids

We determined nucleotide sequences of the 16S rRNA gene of mitochondrial DNA (mtDNA) (about 1.6 kb) for 35 chimpanzee, 13 bonobo, 10 gorilla, 16 orangutan, and 23 gibbon individuals. We compared those data with published sequences and estimated nucleotide diversity for each species. All the ape species showed higher diversity than human. We also constructed phylogenetic trees and networks. The two orangutan subspecies were clearly separated from each other, and Sumatran orangutans showed much higher nucleotide diversity than Bornean orangutans. Some gibbon species did not form monophyletic clusters, and variation within species was not much different from that among species in the subgenus Hylobates.     

The Evolutionary History of the Genus Acanthamoeba and the Identification of Eight New 18S rRNA Gene Sequence Types

ABSTRACT The 18S rRNA gene ( Rns ) phylogeny of Acanthamoeba is being investigated as a basis for improvements in the nomenclature and taxonomy of the genus. We previously analyzed Rns sequences from 18 isolates from morphological groups 2 and 3 and found that they fell into four distinct evolutionary lineages we called sequence types T1-T4. Here, we analyzed sequences from 53 isolates representing 16 species and including 35 new strains. Eight additional lineages (sequence types T5-T12) were identified. Four of the 12 sequence types included strains from more than one nominal species. Thus, sequence types could be equated with species in some cases or with complexes of closely related species in others. The largest complex, sequence type T4, which contained six closely related nominal species, included 24 of 25 keratitis isolates. Rns sequence variation was insufficient for full phylogenetic resolution of branching orders within this complex, but the mixing of species observed at terminal nodes confirmed that traditional classification of isolates has been inconsistent. One solution to this problem would be to equate sequence types and single species. Alternatively, additional molecular information will be required to reliably differentiate species within the complexes. Three sequence types of morphological group 1 species represented the earliest divergence in the history of the genus and, based on their genetic distinctiveness, are candidates for reclassification as one or more novel genera.