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.     

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.     

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.     

Human proliferative sparganosis update

Proliferative sparganosis is one of the most bizarre and mysterious parasitic diseases ever described. The causative parasite is Sparganum proliferum, which is a pseudophyllidean cestode distinct from Spirometra tapeworms. Here we overview this rare but fascinating disease with the all original case reports on human patients published in the last 115 years.Proliferative sparganosis is clearly divided into two disease types, cutaneous and internal proliferative sparganosis. Cutaneous type starts with a skin eruption caused by the dermal invasion of a sparganum. Skin lesion progresses to larger areas of the body if left untreated. Various internal organs and body wall can be eventually affected. The clinical symptoms of patients in this group are very similar to each other. Molecular data suggest that cutaneous proliferative sparganosis is caused by S. proliferum of which genetic variation is limited, regardless of the time or localities of the emergence of patients.Internal proliferative sparganosis, on the other hand, is much more heterogeneous. Some cases show aggressive infection in internal organs, while others show only restricted lesions. Some of the cases that had been cited as proliferative sparganosis in the past literature were removed from the list, because they were judged as cyclophyllidean tapeworm infections. DNA sequencing is mandatory for the definite diagnosis of proliferative sparganosis.The Venezuelan strain of S. proliferum is maintained in experimental mice in Japan, which is fully prepared for the experimental study with advanced technologies in modern molecular biology.     

Intramuscular Sparganosis in the Gastrocnemius Muscle: A Case Report

Sparganosis is a parasitic infection caused by the plerocercoid tapeworm larva of the genus Spirometra. Although the destination of the larva is often a tissue or muscle in the chest, abdominal wall, extremities, eyes, brain, urinary tract, spinal canal, and scrotum, intramuscular sparganosis is uncommon and therefore is difficult to distinguish from a soft tissue tumor. We report a case of intramuscular sparganosis involving the gastrocnemius muscle in an elderly patient who was diagnosed using ultrasonography and MRI and treated by surgical excision. At approximately 1 cm near the schwannoma at the right distal sciatic nerve, several spargana worms were detected and removed.     

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.     

Sources of error in neurotransmitter analysis

A compound having fluorescence characteristics similar to those of 5-hydroxytryptamine (5-HT), when reacted with ninhydrin, is present in several invertebrates (Fasciola hepatica, Aplysia californica, Tritonia diomedia, and Hirudo medicinalis). However, this substance is not identical with 5-HT [ Andreini, G. C., Beretta, C., Faustini, R., and Gallina, G. (1970)Experientia26, 166–167]. We confirmed these findings for Fasciola and also observed this substance in Spirometra mansonoides and mouse brain. Using chromatography and amino acid analysis we identified this substance as lysine. While 5-HT is also demonstrable in Spirometra, no 5-HT is present in Fasciola. Although epinephrine and dopamine seem to be present in Spirometra when the hydroxyindole technique of Laverty and Taylor [ Laverty, R., and Taylor, K. M. (1968)Anal. Biochem.22, 269–279] is used, neither amine is detectable in adult Spirometra by mass spectrometric analysis. A relatively high concentration of tyrosine in Spirometra could account for the apparent presence of dopamine. Therefore, lysine, tyrosine, and possibly other substances can be sources of error in the detection of biogenic amines in invertebrates.     

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).     

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 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.     

First Clinical Cases of Spirometrosis in Two Cats in Korea

This study reports the first two clinical cases of spirometrosis caused by Spirometra sp. in cats in Korea. In these two cases, the cats vomited, and long proglottids of tapeworm were recovered. The sick cats presented with anorexia and lethargy. However, they unexpectedly showed no diarrhea, which is the main symptom of spirometrosis. Based on a fecal floatation test as well as morphological and molecular analyses, the parasite was diagnosed as Spirometra sp. The 2 cases were treated with praziquantel. This study suggests regular monitoring of health and deworming in companion animals, even when animals are well cared for, with regular preventive medication. Additionally, spirometrosis should be considered in the differential diagnosis in cases of gastrointestinal symptoms in Spirometra endemic areas.     

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.     

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.     

Relationship of species composition of tropical seagrass meadows to multiple physical environmental factors

The purpose of this study was to examine the relationship between species composition of tropical seagrasses and various physical environmental factors: depth, sediment thickness and silt–clay content in the sediments. We investigated species composition and abundance of seagrasses as well as the physical environmental factors for six transects around Ishigaki Island, southwest Japan. Eight species occurred in the quadrat census. The occurrence frequencies ranged from 66.8% (Thalassia hemprichii) to 4.5% (Enhalus acoroides). Both canonical correspondence analysis (CCA) and cluster analysis elucidated that depth was mainly responsible for the distributions of species and assemblage type. Monte Carlo permutation for partial CCA revealed that 37.5% of the variance was explained by depth, 10.3% by sediment thickness and 4.6% by silt–clay content in the sediment. Twenty-six sites were categorized into four assemblage types by a cluster analysis using the leaf area index (LAI; the ratio of total leaf area to bottom area) as a measure of species abundance. Type I was dominated by T. hemprichii and Cymodocea rotundata, Type II by C. serrulata, Type III by E. acoroides, and Type IV by Halodule pinifolia and Halophila ovalis. Type I occurred mostly in the intertidal zone (91.3±30.5 cm below MSL, mean sea level), Type II in the subtidal zone (179.1±75.0 cm below MSL) and Type IV in both shallow sites (between 47.8 and 75.6 cm below MSL) and in those with low silt–clay contents (between 2.0 and 3.8%).     

Anisakis simplex and Anisakis physeteris: physicochemical properties of larval and adult hemoglobins

To resolve the taxonomic relationship between two types of parasitic nematode larvae (Type I and II) and two species of parasitic nematode adults (Anisakis simplex and A. physeteris) of the aquatic ascarid genus Anisakis, collected in Japanese coastal water, a comparison was made of their hemoglobins' physicochemical properties. The larval hemoglobins were more similar to each other in electrophoretic pattern than to either adult, indeed there were few similarities whatsoever in these patterns of larval and adult hemoglobins. However, isoelectric points were 6.2 for the Type I larva and for A. simplex and 5.4 for the Type II larva and for A. physeteris. All samples showed identical patterns in spectrophotometric scanning. The circular dichroic spectra of the samples were also virtually identical, although slight differences were noted in the oxygenated hemoglobins; the Type II larva and A. physeteris exhibited a small positive peak at 575 nm but the Type I larva and A. simplex exhibited a much smaller peak (negative position). The sedimentation coefficients of the samples possessed essentially identical values (11.2–12.4). The molecular weights of the samples were estimated, roughly, to be in the range 33 to 43 × 10⁴ by thin-layer chromatography on Sephadex G-200. The evidence suggests that a relationship may exist between the Type I larva and A. simplex, and between the Type II larva and A. physeteris.     

The Bacteriohopanepolyol Inventory of Novel Aerobic Methane Oxidising Bacteria Reveals New Biomarker Signatures of Aerobic Methanotrophy in Marine Systems

Aerobic methane oxidation (AMO) is one of the primary biologic pathways regulating the amount of methane (CH₄) released into the environment. AMO acts as a sink of CH₄, converting it into carbon dioxide before it reaches the atmosphere. It is of interest for (paleo)climate and carbon cycling studies to identify lipid biomarkers that can be used to trace AMO events, especially at times when the role of methane in the carbon cycle was more pronounced than today. AMO bacteria are known to synthesise bacteriohopanepolyol (BHP) lipids. Preliminary evidence pointed towards 35-aminobacteriohopane-30,31,32,33,34-pentol (aminopentol) being a characteristic biomarker for Type I methanotrophs. Here, the BHP compositions were examined for species of the recently described novel Type I methanotroph bacterial genera Methylomarinum and Methylomarinovum, as well as for a novel species of a Type I Methylomicrobium. Aminopentol was the most abundant BHP only in Methylomarinovum caldicuralii, while Methylomicrobium did not produce aminopentol at all. In addition to the expected regular aminotriol and aminotetrol BHPs, novel structures tentatively identified as methylcarbamate lipids related to C-35 amino-BHPs (MC-BHPs) were found to be synthesised in significant amounts by some AMO cultures. Subsequently, sediments and authigenic carbonates from methane-influenced marine environments were analysed. Most samples also did not contain significant amounts of aminopentol, indicating that aminopentol is not a useful biomarker for marine aerobic methanotophic bacteria. However, the BHP composition of the marine samples do point toward the novel MC-BHPs components being potential new biomarkers for AMO.     

Characterization of Spirometra erinaceieuropaei Plerocercoid Cysteine Protease and Potential Application for Serodiagnosis of Sparganosis

BackgroundSparganosis is a neglected but important food-borne parasitic zoonosis. Clinical diagnosis of sparganosis is difficult because there are no specific manifestations. ELISA using plerocercoid crude or excretory–secretory (ES) antigens has high sensitivity but has cross-reactions with other helminthiases. The aim of this study was to characterize Spirometra erinaceieuropaei cysteine protease (SeCP) and to evaluate its potential application for serodiagnosis of sparganosis.ConclusionsThe rSeCP had the CP enzymatic activity and SeCP seems to be important for the survival of plerocercoids in host. The rSeCP is a potential diagnostic antigen for sparganosis.     

Overproduction,purification and characterisation of Tbj1, a novel Type III Hsp40 from Trypanosoma brucei,the African sleeping sickness parasite

The heat shock protein 40 (Hsp40) family of proteins act as co-chaperones of the heat shock protein 70 (Hsp70) chaperone family, and together they play a vital role in the maintenance of cellular homeostasis. The Type III class of Hsp40s are diverse in terms of both sequence identity and function and have not been extensively characterised. The Trypanosoma brucei parasite is the causative agent of Human African Trypanosomiasis, and possesses an unusually large Hsp40 complement, consisting mostly of Type III Hsp40s. A novel T. brucei Type III Hsp40, Tbj1, was heterologously expressed, purified, and found to exist as a compact monomer in solution. Using polyclonal antibodies to the full-length recombinant protein, Tbj1 was found by Western analysis to be expressed in the T. brucei bloodstream-form. Tbj1 was found to be able to assist two different Hsp70 proteins in the suppression of protein aggregation in vitro, despite being unable to stimulate their ATPase activity. This indicated that while Tbj1 did not possess independent chaperone activity, it potentially functioned as a novel co-chaperone of Hsp70 in T. brucei.     

Comparative Genome Analyses of Serratia marcescens FS14 Reveals Its High Antagonistic Potential

S. marcescens FS14 was isolated from an Atractylodes macrocephala Koidz plant that was infected by Fusarium oxysporum and showed symptoms of root rot. With the completion of the genome sequence of FS14, the first comprehensive comparative-genomic analysis of the Serratia genus was performed. Pan-genome and COG analyses showed that the majority of the conserved core genes are involved in basic cellular functions, while genomic factors such as prophages contribute considerably to genome diversity. Additionally, a Type I restriction-modification system, a Type III secretion system and tellurium resistance genes are found in only some Serratia species. Comparative analysis further identified that S. marcescens FS14 possesses multiple mechanisms for antagonism against other microorganisms, including the production of prodigiosin, bacteriocins, and multi-antibiotic resistant determinants as well as chitinases. The presence of two evolutionarily distinct Type VI secretion systems (T6SSs) in FS14 may provide further competitive advantages for FS14 against other microbes. To our knowledge, this is the first report of comparative analysis on T6SSs in the genus, which identifies four types of T6SSs in Serratia spp.. Competition bioassays of FS14 against the vital plant pathogenic bacterium Ralstonia solanacearum and fungi Fusarium oxysporum and Sclerotinia sclerotiorum were performed to support our genomic analyses, in which FS14 demonstrated high antagonistic activities against both bacterial and fungal phytopathogens.