Morphological and Molecular Identification of Spirometra Tapeworms (Cestoda: Diphyllobothriidae) from Carnivorous Mammals in the Serengeti and Selous Ecosystems of Tanzania

Spirometra tapeworms (Cestoda: Diphyllobothriidae) collected from carnivorous mammals in Tanzania were identified by the DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and internal transcribed spacer 1 (ITS1), and by morphological characteristics. A total of 15 adult worms were collected from stool samples and carcasses of Panthera leo, Panthera pardus, and Crocuta crocuta in the Serengeti and Selous ecosystems of Tanzania. Three Spirometra species: S. theileri, S. ranarum and S. erinaceieuropaei were identified based on morphological features. Partial cox1 sequences (400 bp) of 10 specimens were revealed. Eight specimens showed 99.5% similarity with Spirometra theileri ({"type":"entrez-nucleotide","attrs":{"text":"MK955901","term_id":"1796114604","term_text":"MK955901"}}MK955901), 1 specimen showed 99.5% similarity with the Korean S. erinaceieuropaei and 1 specimen had 99.5% similarity with Myanmar S. ranarum. Sequence homology estimates for the ITS1 region of S. theileri were 89.8% with S. erinaceieuropaei, 82.5% with S. decipiens, and 78.3% with S. ranarum; and 94.4% homology was observed between S. decipiens and S. ranarum. Phylogenetic analyses were performed with 4 species of Spirometra and 2 species of Dibothriocephalus (=Diphyllobothrium). By both ML and BI methods, cox1 and ITS1 gave well supported, congruent trees topology of S. erinaceieuropaei and S. theileri with S. decipiens and S. ranarum forming a clade. The Dibothriocephalus species were sisters of each other and collectively forming successive outgroups. Our findings confirmed that 3 Spirometra species (S. theileri, S. ranarum, and S. erinaceieuropaei) are distributed in the Serengeti and Selous ecosystems of Tanzania.     

Mitochondrial Genome of Spirometra theileri Compared with Other Spirometra Species

This study was carried out to provide information on the taxonomic classification and analysis of mitochondrial genomes of Spirometra theileri. One strobila of S. theileri was collected from the intestine of an African leopard (Panthera pardus) in the Maswa Game Reserve, Tanzania. The complete mtDNA sequence of S. theileri was 13,685 bp encoding 36 genes including 12 protein genes, 22 tRNAs and 2 rRNAs with absence of atp8. Divergences of 12 protein-coding genes were as follow: 14.9% between S. theileri and S. erinaceieuropaei, 14.7% between S. theileri and S. decipiens, and 14.5% between S. theileri with S. ranarum. Divergences of 12 proteins of S. theileri and S. erinaceieuropaei ranged from 2.3% in cox1 to 15.7% in nad5, while S. theileri varied from S. decipiens and S. ranarum by 1.3% in cox1 to 15.7% in nad3. Phylogenetic relationship of S. theileri with eucestodes inferred using the maximum likelihood and Bayesian inferences exhibited identical tree topologies. A clade composed of S. decipiens and S. ranarum formed a sister species to S. erinaceieuropaei, and S. theileri formed a sister species to all species in this clade. Within the diphyllobothridean clade, Dibothriocephalus, Diphyllobothrium and Spirometra formed a monophyletic group, and sister genera were well supported.     

Human Infections with Spirometra decipiens Plerocercoids Identified by Morphologic and Genetic Analyses in Korea

Tapeworms of the genus Spirometra are pseudophyllidean cestodes endemic in Korea. At present, it is unclear which Spirometra species are responsible for causing human infections, and little information is available on the epidemiological profiles of Spirometra species infecting humans in Korea. Between 1979 and 2009, a total of 50 spargana from human patients and 2 adult specimens obtained from experimentally infected carnivorous animals were analyzed according to genetic and taxonomic criteria and classified as Spirometra erinaceieuropaei or Spirometra decipiens depending on the morphology. Morphologically, S. erinaceieuropaei and S. decipiens are different in that the spirally coiled uterus in S. erinaceieuropaei has 5-7 complete coils, while in S. decipiens it has only 4.5 coils. In addition, there is a 9.3% (146/1,566) sequence different between S. erinaceieuropaei and S. decipiens in the cox1 gene. Partial cox1 sequences (390 bp) from 35 Korean isolates showed 99.4% (388/390) similarity with the reference sequence of S. erinaceieuropaei from Korea (G1724; GenBank {"type":"entrez-nucleotide","attrs":{"text":"KJ599680","term_id":"646228164","term_text":"KJ599680"}}KJ599680) and an additional 15 Korean isolates revealed 99.2% (387/390) similarity with the reference sequences of S. decipiens from Korea (G1657; GenBank {"type":"entrez-nucleotide","attrs":{"text":"KJ599679","term_id":"646228151","term_text":"KJ599679"}}KJ599679). Based on morphologic and molecular databases, the estimated population ratio of S. erinaceieuropaei to S. decipiens was 35: 15. Our results indicate that both S. erinaceieuropaei and S. decipiens found in Korea infect humans, with S. erinaceieuropaei being 2 times more prevalent than S. decipiens. This study is the first to report human sparganosis caused by S. decipiens in humans in Korea.     

Morphological observation and molecular phylogeny of Spirometra decipiens complex 1 (Cestoda: Diphyllobothriidae) found in cat from Chile

The systematics of tapeworms in the genus Spirometra has been progressing with the accumulation of molecular genetics data, but the taxonomic status of many nominal species remains under debate. We report morphological and molecular-phylogenetic data for a Spirometra species collected from a domestic cat (Felis silvestris catus) in Chiloé Island, Chile. The Spirometra species was shown to be genetically conspecific with Spirometra decipiens complex 1 found in a Pampas fox (Lycalopex gymnocercus) from Argentina, and was closely related to a Hoary fox (Lycalopex vetulus) and rattlesnake (Crotalus durissus) from Brazil. Therefore, the presence of S. decipiens complex 1 was molecularly confirmed for the first time in Chile. The findings of the present study add useful information for the systematics of poorly known Spirometra species in South America.     

A case of human breast sparganosis diagnosed as Spirometra Type I by molecular analysis in Japan

A 92-year-old Japanese woman presented with a mass in the left breast, and sparganosis was suspected by biopsy of the mass. The mass disappeared once, but it reappeared at the same site one year later. For a definitive diagnosis, the mass was surgically removed, and a sparganum-like worm was detected. The causative agent was confirmed as Spirometra Type I (most probably Spirometra mansoni) by mitochondrial DNA analysis. The serological examination also proved the case as sparganosis. Considering the presence of two Spirometra species (Type I and II) in Asia, particularly Japan, molecular analysis of the causative agents is highly recommended to understand the epidemiology, infection sources, and pathogenicity in humans in both species, if the parasite specimens are available.     

Genetic Identification of Spirometra decipiens Plerocercoids in Terrestrial Snakes from Korea and China

Human sparganosis is a zoonotic disease caused by infection with larval forms (procercoid/plerocercoid) of Spirometra spp. The purpose of this study was to identify Spirometra spp. of infected snakes using a multiplex PCR assay and phylogenetic analysis of mitochondrial DNA sequence data from the spargana of terrestrial snakes obtained from Korea and China. A total of 283 snakes were obtained that included 4 species of Colubridae comprising Rhabdophis tigrinus tigrinus (n=150), Dinodon rufozonatum rufozonatum (n=64), Elaphe davidi (n=2), and Elaphe schrenkii (n=7), and 1 species of Viperidae, Agkistrodon saxatilis (n=60). The snakes were collected from the provinces of Chungbuk, Chungnam, and Gyeongbuk in Korea (n=161), and from China (n=122). The overall infection rate with spargana was 83% (235/283). The highest was recorded for D. rufozonatum rufozonatum (100%), followed by A. saxatilis (85%) and R. tigrinus tigrinus (80%), with a negative result for E. davidi (0%) and E. schrenkii (0%). The sequence identities between the spargana from snakes (n=50) and Spirometra erinaceieuropaei ({"type":"entrez-nucleotide","attrs":{"text":"KJ599680","term_id":"646228164","term_text":"KJ599680"}}KJ599680) or S. decipiens ({"type":"entrez-nucleotide","attrs":{"text":"KJ599679","term_id":"646228151","term_text":"KJ599679"}}KJ599679) control specimens were 90.8% and 99.2%, respectively. Pairwise genetic distances between spargana (n=50) and S. decipiens ranged from 0.0080 to 0.0107, while those between spargana and S. erinaceieuropaei ranged from 0.1070 to 0.1096. In this study, all of the 904 spargana analyzed were identified as S. decipiens either by a multiplex PCR assay (n=854) or mitochondrial cox1 sequence analysis (n=50).     

Genetic diversity of two mitochondrial DNA genes in Spirometra erinaceieuropaei (Cestoda: Diphyllobothridae) from Poland

The tapeworm species Spirometra erinaceieuropaei was documented mainly in Asia and Europe. In recent years, plerocercoid larvae (spargana) of this parasite have been found in different hosts in north‐eastern Poland. The evolutionary history and way of S. erinaceieuropaei spreading across Eurasia have been not described yet. However, this phenomenon could be closely related to the evolutionary history and migration routes of studied tapeworm host species. We investigated the genetic variability and divergence pattern among S. erinaceieuropaei populations in intermediate and paratenic hosts from north‐eastern Poland based on complete mitochondrial sequences of cytochrome b (cytb) and cytochrome c oxidase subunit I (cox1) genes. Analysis of 319 consolidated sequences of these two genes showed no genetic structure across study area. Comparison of sequences from Poland and China showed distinct separation of S. erinaceieuropaei populations from these two regions. They split from their common ancestor approximately 28.6 million years ago. Demographic expansion of Polish population of S. erinaceieuropaei started from glacial refugia approximately 12.5 thousand years ago, and recent population expansion has been observed in the tapeworm population from north‐eastern Poland.     

Proteomic and Immunological Identification of Diagnostic Antigens from Spirometra erinaceieuropaei Plerocercoid

Human sparganosis is a food-borne parasitic disease caused by the plerocercoids of Spirometra species. Clinical diagnosis of sparganosis is crucial for effective treatment, thus it is important to identify sensitive and specific antigens of plerocercoids. The aim of the current study was to identify and characterize the immunogenic proteins of Spirometra erinaceieuropaei plerocercoids that were recognized by patient sera. Crude soluble extract of the plerocercoids were separated using 2-dimensional gel electrophoresis coupled with immunoblot and mass spectrometry analysis. Based on immunoblotting patterns and mass spectrometry results, 8 antigenic proteins were identified from the plerocercoid. Among the proteins, cysteine protease protein might be developed as an antigen for diagnosis of sparganosis.     

Sparganosis Presenting as Cauda Equina Syndrome with Molecular Identification of the Parasite in Tissue Sections

A 52-year-old woman presented with lower back pain, progressive symmetrical paraparesis with sensory impairment, and sphincter disturbance. Magnetic resonance imaging (MRI) of the whole spine revealed multiple intradural extramedullary serpiginous-mass lesions in the subarachnoid space continuously from the prepontine to the anterior part of the medulla oblongata levels, C7, T2-T8, and T12 vertebral levels distally until the end of the theca sac and filling-in the right S1 neural foramen. Sparganosis was diagnosed by demonstration of the sparganum in histopathological sections of surgically resected tissues and also by the presence of serum IgG antibodies by ELISA. DNA was extracted from unstained tissue sections, and a partial fragment of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using a primer set specific for Spirometra spp. cox1. After sequencing of the PCR-amplicon and alignment of the nucleotide sequence data, the causative agent was identified as the larva of Spirometra erinaceieuropaei.     

The first report of sparganosis (Spirometra sp.) in Eurasian badger (Meles meles)

Spirometra sp. is a diphyllobothriid cestode which reproduces mainly in cat-like carnivores and canids. Several animal species that are not definitive hosts function as paratenic hosts, in which plerocercoids migrate to other tissues causing sparganosis. In this paper we describe the first case of sparganosis (Spirometra sp.) in Eurasian badger (Meles meles). It was found in an adult female Eurasian badger killed on the road in the Białowieża Primeval Forest (north-eastern Poland) in April 2013. At necropsy, 128 complete and 40 fragments of plerocercoids (spargana) were found and were located subcutaneously, mainly on the hind legs and along the spine. The average length of spargana was 87 ± 38 mm. No adult Spirometra sp. tapeworms were found in the animal intestine, indicating that the investigated badger was a paratenic host for Spirometra sp. Analysis of 18S rRNA gene sequences combined with morphological examination confirmed affiliation of the concerned plerocercoids to genus Spirometra.     

The nucleotide sequence and predicted secondary structure of small subunit (18S) ribosomal RNA from Spirometra erinaceieuropaei

The nucleotide (nt) sequence of a small subunit (18S) ribosomal RNA gene from the plerocercoid of Spirometra erinaceieuropaei (SEP) was determined. The gene with 2182 bp in length is larger than that of most eukaryotes. Extra nt sequences occur in regions known to be variable (V4 and V7). The predicted secondary structure of the nt positions 679–933 (V4) revealed different helices from that of other eukaryotes. The region between nt positions 1540 and 1749 (V7) was different from that of other eukaryotes, but the secondary structure prediction by computer analysis demonstrated that this part of 18S rRNA sequence from S. erinaceieuropaei may form a single extended helix. Nt that were aligned with those of nine other parasites were used to estimate phylogenetic relationships. The data presented here clearly indicate that S. erinaceieuropaei is closely related to Echinococcus granulosus.     

Microbial contributions to bronchial asthma occurrence in children: A metagenomic study

Bronchial asthma, a common chronic respiratory disease in children, is traditionally regarded as a noninfectious disease. Current hypotheses, however, argue that asthma can be caused by microbial infection. We, therefore, hypothesize that a variety of microbes are more commonly found in the sputum of children with asthma, and these microbes may contribute to the occurrence and development of asthma. The present study proposes to use metagenomic approach to explore microbial diversity and to identify the microbial community characteristics of sputum from children with asthma. We found that microbial communities in the sputum of children differed significantly between asthmatics and controls. Kruskal-Wallis testing showed that 16 phyla, 104 genera, and 159 species were significantly downregulated, whereas two phyla including Platyhelminthes phylum and Chordata phylum, two genera including Spirometra genus and Homo sapiens, and the Spirometra erinaceieuropaei species were significantly upregulated in asthma patients compared with controls (P < 0.05). Among them, H. sapiens and S. erinaceieuropaei exhibited 2.3- and 2.0-fold overabundance in asthmatics vs controls, respectively. Meanwhile, metastats assay demonstrated that 31 phyla, 400 genera, and 813 species were significantly downregulated, whereas two phyla, 10 genera, and 16 species were significantly upregulated in asthma patients compared with controls (P < 0.05). Among them, Tetrahymena thermophila and Candidatus Zinderia insecticola exhibited 4.7-fold overabundance in asthmatics vs controls. Our study establishes a link between microbial infection and the mechanisms leading to asthma development, which will be useful for developing novel diagnostic biomarkers and aiding in the prevention and control of asthma.     

Genetic Structure Analysis of Spirometra erinaceieuropaei Isolates from Central and Southern China

Background

Sparganosis caused by invasion of the plerocercoid larvae (spargana) of Spirometra erinaceieuropaei have increased in recent years in China. However, the population genetic structure regarding this parasite is still unclear. In this study, we used the sequences of two mitochondrial genes cytochrome b (cytb) and cytochrome c oxidase subunit I (cox1) to analyze genetic variation and phylogeographic structure of the S. erinaceieuropaei populations.

Methodology/Principal Findings

A total of 88 S. erinaceieuropaei isolates were collected from naturally infected frogs in 14 geographical locations of China. The complete cytb and cox1 genes of each sample was amplified and sequenced. Total 61 haplotypes were found in these 88 concatenated sequences. Each sampled population and the total population have high haplotype diversity (Hd), accompanied by very low nucleotide diversity (Pi). Phylogenetic analyses of haplotypes revealed two distinct clades (HeN+HuN+GZ-AS clade and GX+HN+GZ-GY clade) corresponding two sub-networks yielded by the median-joining network. Pairwise F _ST values supported great genetic differentiation between S. erinaceieuropaei populations. Both negative Fu’s F _S value of neutrality tests and unimodal curve of mismatch distribution analyses supported demographic population expansion in the HeN+HuN+GZ-AS clade. The BEAST analysis showed that the divergence time between the two clades took place in the early Pleistocene (1.16 Myr), and by Bayesian skyline plot (BSP) an expansion occurred after about 0.3 Myr ago.

Conclusions

S. erinaceieuropaei from central and southern China has significant phylogeographic structure, and climatic oscillations during glacial periods in the Quaternary may affect the demography and diversification of this species.     

Multigene phylogenetic analysis reveals non-monophyly of Anisakis s.l. and Pseudoterranova (Nematoda: Anisakidae)

The nematode genera Anisakis s.l. and Pseudoterranova (Anisakidae) include causative agents of anisakiasis and pseudoterranovosis, parasitic diseases resulting from eating undercooked or raw fish or squid. Species in both genera have thus attracted considerable attention especially in public health and taxonomic studies. The phylogenetic relationships of these genera within the subfamily Anisakinae, however, remain to be investigated with dense taxonomic sampling. In this study, we collected an anisakid third-stage larva, and identified it morphologically and molecularly as Pseudoterranova ceticola. Phylogeny of 15 anisakine species, including the newly collected specimen of Ps. ceticola, was reconstructed based on sequences of three mitochondrial (cox1, cox2, and 12S rRNA) and two nuclear (ITS and 28S rRNA) regions. The obtained tree suggested the non-monophyly of Anisakis s.l. and Pseudoterranova. Anisakis s.l. was divided into two groups, which are distinguished from each other by the shape of the ventriculus. Based on phylogenetic relationships and morphology, three species with a shorter ventriculus (“A.” brevispiculata, “A.” paggiae, and “A.” physeteris) were assigned to the genus Skrjabinisakis, as recently proposed. Pseudoterranova ceticola was distantly related to the monophyletic Ps. decipiens species complex. Although the phylogenetic position of the type species Ps. kogiae has not been investigated due to a lack of sequence data, this species may morphologically and ecologically resemble Ps. ceticola, inferring a close kinship between the two species.     

Genetic diversity and geographic distribution of Bemisia tabaci species complex in Nepal

Bemisia tabaci species complex comprises at least 44 cryptic species worldwide. Here, we analyze the nucleotide sequences of mitochondrial cytochrome oxidase subunit 1 (COI) gene obtained from 76 samples of B. tabaci collected from 23 districts in Nepal. This is the first genetic and geographic study of B. tabaci species complex in Nepal. Our phylogenetic study identified the presence of three cryptic species—Asia I, Asia II 1, and Asia II 5—with high interspecific but low intraspecific variations. Among the three cryptic species, Asia II 5 was the most prevalent in Nepal, constituting 64.47% of all the sequenced samples. Based on haplotype network analysis of COI sequences, Asia II 1 was more genetically diversified than the other two cryptic species. Our results provided useful information on the genetic diversity and geographic distribution of B. tabaci in Nepal, which help monitor population changes of B. tabaci at cryptic species level and develop sustainable management strategies for its control.     

A Morphological and Molecular Perspective of Trichoderma viride: Is It One or Two Species?

Trichoderma (Ascomycetes, Hypocreales) strains that have warted conidia are traditionally identified as T. viride, the type species of Trichoderma. However, two morphologically distinct types of conidial warts (I and II) have been found. Because each type corresponds to a unique mitochondrial DNA pattern, it has been questioned whether T. viride comprises more than one species. Combined molecular data (sequences of the internal transcribed spacer 1 [ITS-1] and ITS-2 regions and of part of the 28S rRNA gene along with results of restriction fragment length polymorphism analysis of the endochitinase gene and PCR fingerprinting), morphology, physiology, and colony characteristics distinguish type I and type II as different species. Type I corresponds to “true” T. viride, the anamorph of Hypocrea rufa. Type II represents a new species, T. asperellum, which is, in terms of molecular characteristics, close to the neotype of T. hamatum.     

Hirticrusta gen. nov. segregated from Neofomitella in Polyporaceae (Polyporales)

Taxonomic studies including morphological observations and phylogenetic analyses were conducted on Japanese “uragin-take”, an unidentified species from Amazonia, Brazil and their allies. Phylogenetic analyses using ITS, nrLSU and RPB2 regions revealed that “uragin-take”, Neofomitella polyzonata and the unidentified species formed a monophyletic clade separate from the clade including the other four Neofomitella spp. and that “uragin-take” is conspecific with N. polyzonata. Morphological investigations on authentic specimens revealed that Polyporus subradiatus is a prior name for N. polyzonata. We propose Hirticrusta gen. nov. typified by H. subradiata segregated from Neofomitella, and we erected H. amazonica sp. nov. for the unidentified species. Hirticrusta is characterized by annual to biennial and sessile basidiocarps, semicircular to dimidiate pileus, velutinous to tomentose hairs on pileus surface, buff to brown context with a crustose layer indicated by a dark brown line forming a longitudinal section below the superficial hairs, a trimitic hyphal system, crustose layer composed of parallel and densely arranged brown hyphae and cylindrical basidiospores. The new species, H. amazonica is distinguishable from other polypores by downy and long tomentum on the pileus surface (up to 20 mm thick), brown context with a dark brown layer below the tomentum and round pores (5–7/mm).