Complete sequence and structure of ribosomal RNA gene of Heterosporis anguillarum

The ribosomal RNA (rRNA) gene region of the microsporidium Heterosporis anguillarum has been examined. Complete DNA sequence data (4060 bp, GenBank Accession No. AF402839) of the rRNA gene of H. anguillarum are presented for the small subunit gene (SSU rRNA: 1359 bp), the internal transcribed spacer (ITS: 37 bp), and the large subunit gene (LSU rRNA: 2664 bp). The secondary structures of the H. anguillarum SSU and LSU rRNA genes are constructed and described. This is the first complete sequence of an rRNA gene published for a fish-infecting microsporidian species. In the phylogenetic analysis, the sequences, including partial SSU rRNA, ITS, and partial LSU rRNA sequences of the fish-infecting microsporidia, were aligned and analysed. The taxonomic position of H. anguillarum as suggested by Lom et al. (2000; Dis Aquat Org 43:225-231) is confirmed in this paper.     

Genetic Variations in the Internal Transcribed Spacer and Mitochondrial Small Subunit rRNA Gene of Naegleria spp.

ABSTRACT: Naegleria spp. are widely distributed free-living amebas, but one species in the genus, N. fowleri , causes acute fulminant primary amebic meningoencephalitis in humans and other animals. Thus, it is important to differentiate N. fowleri from the rest in the genus of Naegleria , and to develop tools for the detection of intra-specific genetic variations. In this study, one isolate each of N. australiensis, N. gruberi, N. jadini , and N. lovaniensis and 22 isolates of N. fowleri were characterized at the internal transcribed spacers (ITS) and mitochondrial small subunit rRNA (mtSSU rRNA) gene. The mtSSU rRNA primers designed amplified DNA of all isolates, with distinct sequences obtained from all species examined. In contrast, the ITS primers only amplified DNA from N. lovaniensis and N. fowleri , with minor sequence differences between the two. Three genotypes of N. fowleri were found among the isolates analyzed in both the mtSSU rRNA gene and ITS. The extent of sequence variation was greater in the mtSSU rRNA gene, but the ITS had the advantage of length polymorphism. These data should be useful in the development of molecular tools for rapid species differentiation and genotyping of Naegleria spp.     

Genetic characterization of Strongyloides spp. from captive, semi-captive and wild Bornean orangutans (Pongo pygmaeus) in Central and East Kalimantan, Borneo, Indonesia

Orangutans (Pongo spp.), Asia's only great apes, are threatened in their survival due to habitat loss, hunting and infections. Nematodes of the genus Strongyloides may represent a severe cause of death in wild and captive individuals. In order to better understand which Strongyloides species/subspecies infect orangutans under different conditions, larvae were isolated from fecal material collected in Indonesia from 9 captive, 2 semi-captive and 9 wild individuals, 18 captive groups of Bornean orangutans and from 1 human working with wild orangutans. Genotyping was done at the genomic rDNA locus (part of the 18S rRNA gene and internal transcribed spacer 1, ITS1) by sequencing amplicons. Thirty isolates, including the one from the human, could be identified as S. fuelleborni fuelleborni with 18S rRNA gene identities of 98·5-100%, with a corresponding published sequence. The ITS1 sequences could be determined for 17 of these isolates revealing a huge variability and 2 main clusters without obvious pattern with regard to attributes of the hosts. The ITS1 amplicons of 2 isolates were cloned and sequenced, revealing considerable variability indicative of mixed infections. One isolate from a captive individual was identified as S. stercoralis (18S rRNA) and showed 99% identity (ITS1) with S. stercoralis sequences from geographically distinct locations and host species. The findings are significant with regard to the zoonotic nature of these parasites and might contribute to the conservation of remaining orangutan populations.     

Inter- and intrasporal nuclear ribosomal gene sequence variation within one isolate of arbuscular mycorrhizal fungus, Diversispora sp.

Most molecular ecological studies of arbuscular mycorrhizal fungi (AMF) have been based on the rRNA gene sequences. However, information about intraspecific nucleotide variation is still limited in these fungi. In this study, we calculated the inter- and intrasporal nucleotide variation of Diversispora sp. EE1 using 78 cloned sequences from four spores within a ca 4960 bp fragment of the nuclear ribosomal operon spanning the near full length small ribosomal subunit (SSU) rRNA gene, the full internal transcribed spacer (ITS: ITS1-5.8S-ITS2) and ca 2740 bp of the large ribosomal subunit (LSU) rRNA gene. Data for each marker region (SSU, ITS and LSU) originated from the very same spores. Sequence variation resulting from point mutations and small indels was recorded in all regions. Highest sequence variation was observed in the ITS region at both the inter- and intrasporal levels. The ITS1 component was more variable than ITS2, whilst the 5.8S gene was the least variable component of the ITS region. Evolutionary divergence of gene copies between spores was intermediate for the LSU and lowest for the SSU. The SSU and the LSU genes had relatively similar evolutionary divergence per spore. Sequence variant richness was not exhaustive for any of the marker regions, indicating that multiple sequences per spore from multiple spores are needed when characterizing a species. This study provides reference sequences for ecological studies, permitting identification of AMF using any of the ribosomal regions or primer systems.     

Cryptic diversity of the symbiotic cyanobacterium Synechococcus spongiarum among sponge hosts

Cyanobacteria are common members of sponge-associated bacterial communities and are particularly abundant symbionts of coral reef sponges. The unicellular cyanobacterium Synechococcus spongiarum is the most prevalent photosynthetic symbiont in marine sponges and inhabits taxonomically diverse hosts from tropical and temperate reefs worldwide. Despite the global distribution of S. spongiarum , molecular analyses report low levels of genetic divergence among 16S ribosomal RNA (rRNA) gene sequences from diverse sponge hosts, resulting either from the widespread dispersal ability of these symbionts or the low phylogenetic resolution of a conserved molecular marker. Partial 16S rRNA and entire 16S–23S rRNA internal transcribed spacer (ITS) genes were sequenced from cyanobacteria inhabiting 32 sponges (representing 18 species, six families and four orders) from six geographical regions. ITS phylogenies revealed 12 distinct clades of S. spongiarum that displayed 9% mean sequence divergence among clades and less than 1% sequence divergence within clades. Symbiont clades ranged in specificity from generalists to specialists, with most (10 of 12) clades detected in one or several closely related hosts. Although multiple symbiont clades inhabited some host sponges, symbiont communities appear to be structured by both geography and host phylogeny. In contrast, 16S rRNA sequences were highly conserved, exhibiting less than 1% sequence divergence among symbiont clades. ITS gene sequences displayed much higher variability than 16S rRNA sequences, highlighting the utility of ITS sequences in determining the genetic diversity and host specificity of S. spongiarum populations among reef sponges. The genetic diversity of S. spongiarum revealed by ITS sequences may be correlated with different physiological capabilities and environmental preferences that may generate variable host–symbiont interactions.     

Genetic Diversity of Ostreopsis ovata (Dinophyceae) from Malaysia

The genus Ostreopsis is an important component of benthic and epiphytic dinoflagellate assemblages in coral reefs and seaweed beds of Malaysia. Members of the species may produce toxins that contribute to ciguatera fish poisoning. In this study, two species have been isolated and cultured, Ostreopsis ovata and Ostreopsis lenticularis. Analyses of the 5.8S subunit and internal transcribed spacer regions ITS1 and ITS2 of the ribosomal RNA gene sequences of these two species showed that they are separate species, consistent with morphological designations. The nucleotide sequences of the 5.8S subunit and ITS1 and ITS2 regions of the rRNA gene were also used to evaluate the interpopulation and intrapopulation genetic diversity of O. ovata found in Malaysian waters. Results showed a low level of sequence divergence within populations. At the interpopulation level, the rRNA gene sequence distinguished two groups of genetically distinct strains, representative of a Malacca Straits group (isolates from Port Dickson) and a South China Sea group (isolates from Pulau Redang and Kota Kinabalu). Part of the sequences in the ITS regions may be useful in the design of oligonucleotide probes specific for each group. Results from this study show that the ITS regions can be used as genetic markers for taxonomic, biogeographic, and fine-scale population studies of this species. Received September 15, 2000; accepted December 15, 2000     

Sequence variation in nuclear ribosomal small subunit,internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate‐dependent

Arbuscular mycorrhizal (AM) fungi are known to exhibit high intra‐organism genetic variation. However, information about intra‐ vs. interspecific variation among the genes commonly used in diversity surveys is limited. Here, the nuclear small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene portions were sequenced from 3 to 5 individual spores from each of two isolates of Rhizophagus irregularis and Gigaspora margarita. A total of 1482 Sanger sequences (0.5 Mb) from 239 clones were obtained, spanning ~4370 bp of the ribosomal operon when concatenated. Intrasporal and intra‐isolate sequence variation was high for all three regions even though variant numbers were not exhausted by sequencing 12–40 clones per isolate. Intra‐isolate nucleotide variation levels followed the expected order of ITS > LSU > SSU, but the values were strongly dependent on isolate identity. Single nucleotide polymorphism (SNP) densities over 4 SNP/kb in the ribosomal operon were detected in all four isolates. Automated operational taxonomic unit picking within the sequence set of known identity overestimated species richness with almost all cut‐off levels, markers and isolates. Average intraspecific sequence similarity values were 99%, 96% and 94% for amplicons in SSU, LSU and ITS, respectively. The suitability of the central part of the SSU as a marker for AM fungal community surveys was further supported by its level of nucleotide variation, which is similar to that of the ITS region; its alignability across the entire phylum; its appropriate length for next‐generation sequencing; and its ease of amplification in single‐step PCR.     

Intraspecific and within-isolate sequence variation in the ITS rRNA gene region of Pythium mercuriale sp. nov. (Pythiaceae)

Sixteen Pythium isolates from diverse hosts and locations, which showed similarities in their morphology and sequences of the internal transcribed spacer (ITS) region of their rRNA gene, were investigated. As opposed to the generally accepted view, within single isolates ITS sequence variations were consistently found mostly as part of a tract of identical bases (A-T) within ITS1, and of GT or GTTT repeats within the ITS2 sequence. Thirty-one different ITS sequences obtained from 39 cloned ITS products from the 16 isolates showed high sequence and length polymorphisms within and between isolates. However, in a phylogenetic analysis, they formed a cluster distinct from those of other Pythium species. Additional sequencing of two nuclear genes (elongation factor 1 alpha and beta-tubulin) and one mitochondrial gene (nadh1) revealed high levels of heterozygosity as well as polymorphism within and between isolates, with some isolates possessing two or more alleles for each of the nuclear genes. In contrast to the observed variation in the ITS and other gene areas, all isolates were phenotypically similar. Pythium mercuriale sp. nov. (Pythiaceae) is characterized by forming thin-walled chlamydospores, subglobose to obovoid, papillate sporangia proliferating internally and smooth-walled oogonia surrounded by multiple antheridia. Maximum likelihood phylogenetic analyses based on both ITS and beta-tubulin sequence data place P. mercuriale in a clade between Pythium and Phytophthora.     

Molecular data and phylogeny of Nosema infecting lepidopteran forest defoliators in the genera Choristoneura and Malacosoma

ABSTRACT. Nosema isolates from five lepidopteran forest defoliators, Nosema fumiferanae from spruce budworm, Choristoneura fumiferana ; a Nosema sp. from jack pine budworm, Choristoneura pinus pinus and western spruce budworm, Choristoneura occidentalis ( Nosema sp. CPP and Nosema sp. CO, respectively); Nosema thomsoni from large aspen tortrix, Choristoneura conflictana ; and Nosema disstriae , from the forest tent caterpillar, Malacosoma disstria were compared based on their small subunit (SSU) ribosomal RNA (rRNA) gene sequences. Four of the species sequenced, N. fumiferanae , Nosema sp. CPP, Nosema sp. CO, and N . disstriae have a high SSU rDNA sequence identity (0.6%–1.5%) and are members of the "true Nosema " clade. They all showed the reverse arrangement of the (large subunit [LSU]–internal transcribed spacer [ITS]–SSU) of the rRNA gene. The fifth species, N. thomsoni has the usual (SSU–ITS–LSU) arrangement and is not a member of this clade showing only an 82% sequence similarity. We speculate, therefore, that a genetic reversal may have occurred in the common ancestor to the "true Nosema " clade. Although, the mechanism for rearrangement of the rRNA gene subunits is not known we provide a possible explanation for the localization. N. fumiferanae , Nosema sp. CPP, and Nosema sp. CO clustered together on the inferred phylogenetic tree. The high sequence similarities, the reverse arrangement in the rRNA gene subunits, and the phylogenetic clustering suggest that these three species are closely related but separate species.     

Presence of a novel 16S-23S rRNA gene intergenic spacer insert in Bradyrhizobium canariense strains

Seven slow-growing bacterial strains isolated from root nodules of yellow serradella (Ornithopus compressus) that originated from Asinara Island on North Western Sardinia in Italy were characterized by partial 16S rRNA gene and intergenic spacer (ITS) sequencing as well as amplified fragment length polymorphism (AFLP) genomic fingerprinting. The results indicated that the O. compressus isolates belong to the Bradyrhizobium canariense species. The analysis of ITS sequences divided the branch of B. canariense strains into two statistically separated groups (ITS clusters I and II). All the strains in ITS cluster I showed the presence of unique oligonucleotide insert TTAGAGACTTAGGTTTCTK. This insert was neither found in other described species of the family Rhizobiaceae nor in any other bacterial families and can be used as a natural and high selective genetic marker for ITS cluster I of B. canariense strains. ITS grouping of O. compressus isolates was supported by the unweighted pair group method with arithmetic averages cluster analysis of their AFLP patterns, suggesting that the strains of ITS cluster II were genetically closer to each other than to isolates from the ITS cluster I. A taxonomic importance is supposed of the revealed 19 bp ITS insert for an intraspecific division within high heterogeneous B. canariense species.     

Variation of 16S-23S internally transcribed spacer sequence and intervening sequence in rDNA among the three major Agrobacterium species

We analyzed 16S-23S internally transcribed spacer (ITS) and neighboring sequences among 37 strains belonging to the three major pathogenic Agrobacterium species, in order to know variation in each species and to develop a simple discrimination method. Number of ITS size variation was 9, 4, and 7 in Agrobacterium tumefaciens, Agrobacterium vitis, and Agrobacterium rhizogenes, respectively. The ITS sequence of most strains in each species was distinguishable from that of the other two species. The region surrounded by 16S rRNA gene and trn(Ala) contained information to distinguish between the ITS variants and was easy for sequencing. Intervening sequences (IVSs) in 23S rRNA gene were classified into short and long types in each species. Some long-type IVSs of A. vitis were very similar to that of A. tumefaciens, while the other long-type IVSs of A. vitis were very similar to that of A. rhizogenes. Two A. vitis strains simultaneously contained both types of IVS. Similarly, the two exceptional A. vitis strains possessed A. tumefaciens-type ITS in addition to A. vitis-type ITS. These results suggest horizontal transfer of rDNA and subsequent recombination. Among the three species, A. tumefaciens was most variable based on 16S rRNA gene, ITS and IVS sequences.     

Ribosomal variation in six species of shape Fusarium

Eighteen isolates representing six Fusarium species from diverse hosts and geographical origins were evaluated to determine ribosomal DNA variation using polymerase chain reaction and restriction fragment length polymorphisms. No length variation was observed for amplified 18S and 28S regions. However, amplification of the ITS region showed one isolate, a F. oxysporum, to be about 120 bp larger than the remaining 17. Restriction digestions in the 18S region revealed polymorphisms within species of F. oxysporum and F. solani. An amplified variable stretch of the 28S gene showed restriction site differences between F. avenecum, F. sambucinum and F. sporotrichioides. A large degree of polymorphism was observed both between and within species in the ITS region. Therefore, entire sequences of the ITS and the 5.8S subunit were obtained for 17 of the 18 isolates. These sequences, along with those from eight additional isolates, were analysed using PAUP to assess the occurrence of DNA sequence divergence within the ITS region. The lack of correlation between molecular-based relationships and species affinities inferred from morphology for some isolates indicates that species designation can be unreliable using morphological data alone. Possible reasons for the discordance of the sequence and morphological data are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic heterogeneity in internal transcribed spacer genes of Balantidium coli (Litostomatea, Ciliophora)

The species Balantidium coli is the only ciliate that parasitizes humans. It has been described in other primates, and it has been proposed that the species B. suis from pigs and B. struthionis from ostriches are synonyms of B. coli. Previous genetic analysis of pig and ostrich Balantidium isolates found a genetic polymorphism in the ITS region but its taxonomic relevance was not established. We have extended the genetic analysis to Balantidium isolates of pig, gorilla, human and ostrich origin. We have PCR-amplified and sequenced the ITS region of individual Balantidium cells. The predicted ITS secondary structures of the sequences obtained were transferred by homology modelling to the sequences of other Trichostomatia ciliates (Isotricha, Troglodytella, Lacrymaria and Spathidium) and compared to determine the importance of the differences in the primary sequences. The results show that the ITS2 secondary structure of the species considered follows the general pattern of other ciliates, although with some deviations. There are at least two main types of ITS sequence variants in B. coli which could be present in the same cell and they are common to the mammal and avian hosts studied. These data do not support B. suis and B. struthionis as distinct species.     

Is the trophosome of <Emphasis Type="Italic">Ridgeia piscesae</Emphasis> monoclonal?

The hydrothermal vent tubeworm Ridgeia piscesae relies on intracellular chemolithoautotrophic symbionts for its nutrition. Yet, little is known about symbiont diversity within and between individual worms. We report several lines of molecular evidence for multiple genotypes of very closely related symbionts within the trophosome of the R. piscesae. We examined the distribution of genotypic variants (insertions, deletions, and substitutions) in whole genome shotgun sequences of symbionts from the trophosome of a unique individual R. piscesae and the pooled sequences of five other tubeworms of the same species. Observed heterogeneity is unlikely to be the result of recent point or structural mutations of a monoclonal symbiont lineage. To assess inter-host diversity we examined single nucleotide polymorphisms (SNPs) in pyrosequences of the highly variable regions V1 to V3 of the symbiont 16S rRNA gene across 53 individual hosts from two vent sites. Most of the identified SNPs were found in more than one individual, and one seemed to be region specific. Two of the identified SNPs were also present in metagenomic data generated from high-throughput sequencing of trophosome material from an individual R. piscesae. Finally, we observed compositional and structural variations of CRISPR spacers within a CRISPR array.     

Molecular Characterization of Various Trichomonad Species Isolated from Humans and Related Mammals in Indonesia

Trichomonad species inhabit a variety of vertebrate hosts; however, their potential zoonotic transmission has not been clearly addressed, especially with regard to human infection. Twenty-one strains of trichomonads isolated from humans (5 isolates), pigs (6 isolates), rodents (6 isolates), a water buffalo (1 isolate), a cow (1 isolate), a goat (1 isolate), and a dog (1 isolate) were collected in Indonesia and molecularly characterized. The DNA sequences of the partial 18S small subunit ribosomal RNA (rRNA) gene or 5.8S rRNA gene locus with its flanking regions (internal transcribed spacer region, ITS1 and ITS2) were identified in various trichomonads; Simplicimonas sp., Hexamastix mitis, and Hypotrichomonas sp. from rodents, and Tetratrichomonas sp. and Trichomonas sp. from pigs. All of these species were not detected in humans, whereas Pentatrichomonas hominis was identified in humans, pigs, the dog, the water buffalo, the cow, and the goat. Even when using the high-resolution gene locus of the ITS regions, all P. hominis strains were genetically identical; thus zoonotic transmission between humans and these closely related mammals may be occurring in the area investigated. The detection of Simplicimonas sp. in rodents (Rattus exulans) and P. hominis in water buffalo in this study revealed newly recognized host adaptations and suggested the existence of remaining unrevealed ranges of hosts in the trichomonad species.     

BIOGEOGRAPHIC ANALYSIS OF THE GLOBALLY DISTRIBUTED HARMFUL ALGAL BLOOM SPECIES ALEXANDRIUM MINUTUM (DINOPHYCEAE) BASED ON rRNA GENE SEQUENCES AND MICROSATELLITE MARKERS1

The toxic dinoflagellate Alexandrium minutum Halim is one of three species that comprise the “minutum” species complex. This complex is notable due to its role in the etiology of paralytic shellfish poisoning (PSP). Recent increases in PSP incidence and the geographic expansion of toxin‐producing Alexandrium dinoflagellates have prompted the intensive examination of genetic relationships among globally distributed strains to address questions regarding their present distribution and reasons for their apparent increase. The biogeography of A. minutum was studied using large subunit ribosomal DNA gene (LSU rRNA) and internal transcribed spacer (ITS) sequences and genotypic data from 12 microsatellite loci. rRNA gene and ITS sequencing data distinguished between two clades, herein termed the “Global” and the “Pacific”; however, little to no resolution was seen within each clade. Genotypic data from 12 microsatellite loci provided additional information regarding genetic relationships within the Global clade, but it was not possible to amplify DNA from the Pacific clade using these markers. With the exception of isolates from Italy and Spain, strains generally clustered according to origin, revealing geographic structuring within the Global clade. Additionally, no evidence supported the separation of A. lusitanicum and A. minutum as different species. With the use of microsatellites, it is now possible to initiate studies on the origin, history, and genetic heterogeneity of A. minutum that were not previously possible using only rRNA gene sequence data. This study demonstrates the power of combining a marker with intermediate resolution (rRNA sequences) with finer‐scale markers (microsatellites) to examine intraspecies variability among globally distributed isolates and represents the first effort to employ this technique in A. minutum.     

Phylogenetic validation of the genera Angomonas and Strigomonas of trypanosomatids harboring bacterial endosymbionts with the description of new species of trypanosomatids and of proteobacterial symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia culicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history.     

COMPARISON OF THREE COMMON MOLECULAR TOOLS FOR DISTINGUISHING AMONG GEOGRAPHICALLY SEPARATED CLONES OF THE DIATOM SKELETONEMA MARINOI SARNO ET ZINGONE (BACILLARIOPHYCEAE)1

Skeletonema marinoi Sarno et Zingone is a planktonic marine diatom with a widespread geographic distribution. Different populations of this species may show distinct genetic signatures. We have evaluated the utility of three common molecular methods for distinguishing clones of S. marinoi from different geographic regions. Clonal cultures were isolated from the Canadian west coast, south west Portugal, and the east and west coasts of Sweden. All strains originated from resting stages in sediment. More than 90% of the individually isolated chains grew to densities suitable for DNA extraction. Genetic signatures of clones from each sample location were assessed by sequencing variable domains (D1–D3) of the nuclear large subunit (LSU) rRNA gene and internal transcriber spacer (ITS) (ITS‐1, 5.8S and ITS‐2) regions, and also by analysis of randomly amplified polymorphic DNA patterns. Analysis of molecular variance showed that strains from the four geographic areas were significantly separated by all three methods but that differences among European samples were best resolved by ITS 2 sequences.     

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.     

Identification of Clinically Relevant Fungi and Prototheca Species by rRNA Gene Sequencing and Multilocus PCR Coupled with Electrospray Ionization Mass Spectrometry

Background

Multilocus PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is a new strategy for pathogen identification, but information about its application in fungal identification remains sparse.

Methods

One-hundred and twelve strains and isolates of clinically important fungi and Prototheca species were subjected to both rRNA gene sequencing and PCR/ESI-MS. Three regions of the rRNA gene were used as targets for sequencing: the 5′ end of the large subunit rRNA gene (D1/D2 region), and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). Microbial identification (Micro ID), acquired by combining results of phenotypic methods and rRNA gene sequencing, was used to evaluate the results of PCR/ESI-MS.

Results

For identification of yeasts and filamentous fungi, combined sequencing of the three regions had the best performance (species-level identification rate of 93.8% and 81.8% respectively). The highest species-level identification rate was achieved by sequencing of D1/D2 for yeasts (92.2%) and ITS2 for filamentous fungi (75.8%). The two Prototheca species could be identified to species level by D1/D2 sequencing but not by ITS1 or ITS2. For the 102 strains and isolates within the coverage of PCR/ESI-MS identification, 87.3% (89/102) achieved species-level identification, 100% (89/89) of which were concordant to Micro ID on species/complex level. The species-level identification rates for yeasts and filamentous fungi were 93.9% (62/66) and 75% (27/36) respectively.

Conclusions

rRNA gene sequencing provides accurate identification information, with the best results obtained by a combination of ITS1, ITS2 and D1/D2 sequencing. Our preliminary data indicated that PCR/ESI-MS method also provides a rapid and accurate identification for many clinical relevant fungi.