A human case of Stellantchasmus falcatus infection in Korea

In an attempt to find the worm producing unidentified egg, one minute fluke was collect from a Korean patient after praziquantel administration. The fluke was identified to be Stellantchasmus falcatus by the expulsor. Brackish water fish was suggested to be a probable source of the infection.     

Molecular markers for identification of Stellantchasmus falcatus and a phylogenic study using the HAT-RAPD method

Stellantchasmus falcatus is a minute intestinal fluke in the family Heterophyidae. Metacercariae, the infective stage, were reported in a marine fish, mullet Liza subviridis, and a fresh water fish, Dermogenus pusillus, in Thailand. Adults were found in chicks, rats, cats, and humans. Morphological studies were done for comparing Stellantchasmus sp. worms found in 2 different fish hosts; their shapes and organ arrangements were very similar except for the prepharynx length. Therefore, the present study aimed to compare their DNA fingerprints using the HAT-RAPD method for both types of Stellantchasmus and several other related species. Ten arbitrarily selected primers (OPA-04, OPA-09, OPN-02, OPN-03, OPN-09, OPN-12, OPP-11, OPR-15, OPX-13, and OPAD-01) were used. It was found that OPA-09, OPN-03, and OPAD-01 were able to generate S. falcatus specific fragments in mullets which consisted of 200, 760, and 280 bp, respectively. In addition, the results of morphologic, DNA fingerprinting, and phylogenetic analyses strongly suggest that the fresh water and marine specimens of Stellantchamus may be different species.     

Surface ultrastructure of the adult stage of Acanthotrema felis (Trematoda: Heterophyidae)

The surface ultrastructure of Acanthotrema felis (Trematoda: Heterophyidae) adults, recovered from a kitten experimentally infected with the metacercariae, was observed using a scanning electron microscope. The worm was leaf-like, ventrally concave and covered with scale-like multi-pointed tegumental spines. The spines on the anterior surface were short but broad, and had 10-12 pointed tips. The cytoplasmic processes protruded around the spines, like pockets for the spines. The ventrogenital opening was crescent, or kidney-shaped, and had protuberances with minute spines on its surrounding tegument. The spines on the posterior surface were long, but narrow, with 6-8 pointed tips. The cytoplasmic processes on this tegument were ridge-like, and elevated along the row of the spines. The surface ultrastructure of A. felis is generally similar to that of other heterophyid flukes, but some features are characteristic, and may be of taxonomic and bio-ecological significance.     

Phylogenetic Relationships of a Distinct Species of Globodera from Portugal and Two Punctodera Species

Evolutionary relationships based on ribosomal DNA (rDNA) sequence data for a previously unknown species of Globodera from Portugal, Punctodera chalcoensis from Mexico, and P. punctata from Estonia, plus previously published sequences, support the following relationships: (((Cactodera weissi, G. artemisiae, C. milleri), ((G. sp. Bouro, G. sp. Canha, G. sp. Ladoeiro), ((G. pallida, G. rostochiensis), (P. chalcoensis, P. punctata)))), Heterodera avenae). Globodera sp. from Portugal, which can be confused with potato cyst nematodes by phytosanitary services when the identification is based only on morphological characters, is clearly different based on our molecular data. In addition, the rDNA data show the Globodera sp. to be only distantly related to other European Globodera species that parasitize Asteraceae. Punctodera chalcoensis and P. punctata form a sister clade to the G. pallida + G. rostochiensis clade.     

Mucosal Immune Responses of Mice Experimentally Infected with Pygidiopsis summa (Trematoda: Heterophyidae)

Mucosal immune responses against Pygidiopsis summa (Trematoda: Heterophyidae) infection were studied in ICR mice. Experimental groups consisted of group 1 (uninfected controls), group 2 (infection with 200 metacercariae), and group 3 (immunosuppression with Depo-Medrol and infection with 200 metacercariae). Worms were recovered in the small intestine at days 1, 3, 5, and 7 post-infection (PI). Intestinal intraepithelial lymphocytes (IEL), mast cells, and goblet cells were counted in intestinal tissue sections stained with Giemsa, astra-blue, and periodic acid-Schiff, respectively. Mucosal IgA levels were measured by ELISA. Expulsion of P. summa from the mouse intestine began to occur from days 3-5 PI which sustained until day 7 PI. The worm expulsion was positively correlated with proliferation of IEL, mast cells, goblet cells, and increase of IgA, although in the case of mast cells significant increase was seen only at day 7 PI. Immunosuppression suppressed all these immune effectors and inhibited worm reduction in the intestine until day 7 PI. The results suggested that various immune effectors which include IEL, goblet cells, mast cells, and IgA play roles in regulating the intestinal mucosal immunity of ICR mice against P. summa infection.     

Phylogenetic Relationships Based on Ribosomal DNA Data for Four Species of Cyst Nematodes from Italy and One from Syria

Phylogenetic analysis of new ribosomal DNA (rDNA) data for Heterodera mediterranea, H. hordecalis, H. carotae, and H. fici from Italy and H. ciceri from Syria, along with published data for other species, showed high bootstrap support for the following relationships: (((((H. carotae H. cruciferae) H. goettingiana) (((H. trifolii H. ciceri) H. mediterranea) ((H. avenae H. latipons) H. fici))) (Cactodera betulae H. hordecalis)) (Globodera rostochiensis G. pallida)). The rDNA sequence data were for the two internal transcribed spacers (ITS1 and ITS2) plus the 5.8S gene between them. These inferred relationships support the classic ''''Goettingiana Group'''' of H. carotae, H. cruciferae, and H. goettingiana. A clade comprised of Cactodera betulae and H. hordecalis is only distantly related to the other species in the analysis.     

Phylogenetic Analyses in Dorylaimida Using Data from 2-D Protein Patterns

Data from two-dimensional protein patterns for nine dorylaimid isolates were analyzed using PAUP, a computer program for inferring phylogenies under the principle of maximum parsimony. With a variety of available options, including branch swapping and rooting, essentially the same tree was obtained. When isolates of the genus Labronema were analyzed alone, all trees obtained had the same topology, although tree length varied considerably, depending on whether a hypothetical ancestral taxon was included.     

Phylogenetic relationships and generic delimitation of Eurasian Aster (Asteraceae: Astereae) inferred from ITS, ETS and trnL-F sequence data

Background and Aims

The classification and phylogeny of Eurasian (EA) Aster (Asterinae, Astereae, Asteraceae) remain poorly resolved. Some taxonomists adopt a broad definition of EA Aster, whereas others favour a narrow generic concept. The present study aims to delimit EA Aster sensu stricto (s.s.), elucidate the phylogenetic relationships of EA Aster s.s. and segregate genera.

Methods

The internal and external transcribed spacers of nuclear ribosomal DNA and the plastid DNA trnL-F region were used to reconstruct the phylogeny of EA Aster through maximum parsimony and Bayesian analyses.

Key Results

The analyses strongly support an Aster clade including the genera Sheareria, Rhynchospermum, Kalimeris (excluding Kalimeris longipetiolata), Heteropappus, Miyamayomena, Turczaninowia, Rhinactinidia, eastern Asian Doellingeria, Asterothamnus and Arctogeron. Many well-recognized species of Chinese Aster s.s. lie outside of the Aster clade.

Conclusions

The results reveal that EA Aster s.s. is both paraphyletic and polyphyletic. Sheareria, Rhynchospermum, Kalimeris (excluding K. longipetiolata), Heteropappus, Miyamayomena, Turczaninowia, Rhinactinidia, eastern Asian Doellingeria, Asterothamnus and Arctogeron should be included in Aster, whereas many species of Chinese Aster s.s. should be excluded. The recircumscribed Aster should be divided into two subgenera and nine sections. Kalimeris longipetiolata, Aster batangensis, A. ser. Albescentes, A. series Hersileoides, a two-species group composed of A. senecioides and A. fuscescens, and a six-species group including A. asteroides, should be elevated to generic level. With the Aster clade, they belong to the Australasian lineages. The generic status of Callistephus should be maintained. Whether Galatella (including Crinitina) and Tripolium should remain as genera or be merged into a single genus remains to be determined. In addition, the taxonomic status of A. auriculatus and the A. pycnophyllus–A. panduratus clade remains unresolved, and the systematic position of some segregates of EA Aster requires further study.     

Phylogenetic relationships within Chamaecrista sect. Xerocalyx (Leguminosae, Caesalpinioideae) inferred from the cpDNA trnE-trnT intergenic spacer and nrDNA ITS sequences

Chamaecrista belongs to subtribe Cassiinae (Caesalpinioideae), and it comprises over 330 species, divided into six sections. The section Xerocalyx has been subjected to a profound taxonomic shuffling over the years. Therefore, we conducted a phylogenetic analysis using a cpDNA trnE-trnT intergenic spacer and nrDNA ITS/5.8S sequences from Cassiinae taxa, in an attempt to elucidate the relationships within this section from Chamaecrista. The tree topology was congruent between the two data sets studied in which the monophyly of the genus Chamaecrista was strongly supported. Our analyses reinforce that new sectional boundaries must be defined in the Chamaecrista genus, especially the inclusion of sections Caliciopsis and Xerocalyx in sect. Chamaecrista, considered here paraphyletic. The section Xerocalyx was strongly supported as monophyletic; however, the current data did not show C. ramosa (microphyllous) and C. desvauxii (macrophyllous) and their respective varieties in distinct clades, suggesting that speciation events are still ongoing in these specimens.     

Phylogenetic utility of the nuclear genes AGAMOUS 1 and PHYTOCHROME B in palms (Arecaceae): an example within Bactridinae

Background and Aims

Molecular phylogenetic studies of palms (Arecaceae) have not yet provided a fully resolved phylogeny of the family. There is a need to increase the current set of markers to resolve difficult groups such as the Neotropical subtribe Bactridinae (Arecoideae: Cocoseae). We propose the use of two single-copy nuclear genes as valuable tools for palm phylogenetics.

Methods

New primers were developed for the amplification of the AGAMOUS 1 (AG1) and PHYTOCHROME B (PHYB) genes. For the AGAMOUS gene, the paralogue 1 of Elaeis guineensis (EgAG1) was targeted. The region amplified contained coding sequences between the MIKC K and C MADS-box domains. For the PHYB gene, exon 1 (partial sequence) was first amplified in palm species using published degenerate primers for Poaceae, and then specific palm primers were designed. The two gene portions were sequenced in 22 species of palms representing all genera of Bactridinae, with emphasis on Astrocaryum and Hexopetion, the status of the latter genus still being debated.

Key Results

The new primers designed allow consistent amplification and high-quality sequencing within the palm family. The two loci studied produced more variability than chloroplast loci and equally or less variability than PRK, RPBII and ITS nuclear markers. The phylogenetic structure obtained with AG1 and PHYB genes provides new insights into intergeneric relationships within the Bactridinae and the intrageneric structure of Astrocaryum. The Hexopetion clade was recovered as monophyletic with both markers and was weakly supported as sister to Astrocaryum sensu stricto in the combined analysis. The rare Astrocaryum minus formed a species complex with Astrocaryum gynacanthum. Moreover, both AG1 and PHYB contain a microsatellite that could have further uses in species delimitation and population genetics.

Conclusions

AG1 and PHYB provide additional phylogenetic information within the palm family, and should prove useful in combination with other genes to improve the resolution of palm phylogenies.     

A cytogenetic study on human intestinal trematodes of the genus Metagonimus (Digenea: Heterophyidae) in Korea

In order to analyze chromosome numbers and karyotypes of intestinal trematodes belonging to the genus, Metagonimus, the gonad tissues of M. takahashii, M. miyatai, and M. yokogawai were prepared and examined. The number of bivalents in the first meiotic division of M. takahashii was nine (n = 9). The diploid number of M. miyatai was observed to be eighteen (2n = 18) and their chromosomes consisted of one pair of metacentric, 7 pairs of submetacentric, and one pair of telocentric chromosomes. The diploid number of M. yokogawai was thirty-two (2n = 32) and the chromosome complements were composed of two pairs of metacentric, 11 pairs of submetacentric, and three pairs of subtelocentric chromosomes. These results could be a supporting evidence for the validity of the new species, M. miyatai, distinct from M. yokogawai.     

Foodborne Trematode Metacercariae in Fish from Northern Vietnam and Their Adults Recovered from Experimental Hamsters

The prevalence of foodborne trematode (FBT) metacercariae was investigated in fish from 2 localities of northern Vietnam in 2004-2005. Freshwater fish (9 species) were collected from local markets in Hanoi City (n=76) and Nam Dinh Province (n=79), and were examined for FBT metacercariae using the artificial digestion technique. Adult flukes were obtained from hamsters experimentally infected with the metacercariae at day 8 post-infection. Three (Haplorchis pumilio, Centrocestus formosanus, and Procerovum varium) and 6 (Haplorchis taichui, H. pumilio, C. formosanus, P. varium, Stellantchasmus falcatus, and Heterophyopsis continua) species of FBT metacercariae were detected in the 2 regions, respectively. Overall, among the positive fish species, H. pumilio metacercariae were detected in 104 (80.0%) of 130 fish examined (metacercarial density per infected fish; 64.2). C. formosanus metacercariae were found in 37 (40.2%) of 92 fish (metacercarial density; 14.7). P. varium metacercariae were detected in 19 (63.3%) of 30 fish (Anabas testudineus and Mugil cephalus) (metacercarial density; 247.7). S. falcatus metacercariae were found in all 10 M. cephalus examined (metacercarial density; 84.4). H. continua metacercariae (2 in number) were detected in 1 fish of Coilia lindmani. Morphologic characteristics of the FBT metacercariae and their experimentally obtained adults were described. The results have demonstrated that various FBT species are prevalent in northen parts of Vietnam.     

Surface ultrastructure of Pygidiopsis summa (Digenea: Heterophyidae) adult flukes

A scanning electron microscopic study was performed on the surface ultrastructure of Pygidiopsis summa (Digenea: Heterophyidae) adults. Metacercariae were collected from gills and muscles of mullets (Mugil cephalus) caught in a known endemic area, and adult flukes were harvested from dogs after 8 weeks of experimental infection. The worm was calabash form with its posterior part broader than the anterior part. Tegumental spines were densely distributed over the body surface, except on the suckers and genital apparatus, and around the excretory pore. Well differentiated spines were observed on the anterior half of the body, with 14-16 tips ventrally, and 19-20 tips dorsally. On the oral sucker, three pairs of type I sensory papillae (uni-ciliated knob-like swellings) and one pair of type II sensory papillae (aciliated round-swellings) were observed on the anterior and posterior parts of the lip, respectively. On the lip of the ventral sucker, one pair of type II sensory papillae was distributed only on its posterior part. Sperms were seen emerging from or entering into the genital apparatus. The results showed that the surface ultrastructure of P. summa was unique among the heterophyid trematodes, especially in digitation of tegumental spines and in distribution of sensory papillae on oral and ventral suckers.     

Centrocestus formosanus (Digenea: Heterophyidae) encysted in the freshwater fish, Puntius brevis, from Lao PDR

The metacercariae of Centrocestus formosanus, a minute intestinal trematode of mammals and birds, were detected in the freshwater fish, Puntius brevis, from Vientiane Municipality, Lao PDR. The metacercariae were experimentally fed to mice, and adult flukes were recovered in their small intestines 7 days later. The adult flukes were morphologically characterized by having 32 (rarely 34) circumoral spines arranged in 2 alternative rows, a large bipartite seminal vesicle, an oval-shaped ovary, and an X-shaped excretory bladder. Based on these characters, the adults were identified as Centrocestus formosanus (Nishigori, 1924). The taxonomic significance of C. formosanus, in relation to a closely related species, C. caninus (Leiper, 1913), is briefly discussed. It has been first verified by adult worm recovery that C. formosanus is prevalent in Vientiane areas of Lao PDR, taking the freshwater fish, P. brevis, as a second intermediate host.     

Phylogenetic Analysis of the Hoplolaiminae Inferred from Combined D2 and D3 Expansion Segments of 28S rDNA

DNA sequences of the D2-D3 expansion segments of the 28S gene of ribosomal DNA from 23 taxa of the subfamily Hoplolaiminae were obtained and aligned to infer phylogenetic relationships. The D2 and D3 expansion regions are G-C rich (59.2%), with up to 20.7% genetic divergence between Scutellonema brachyurum and Hoplolaimus concaudajuvencus. Molecular phylogenetic analysis using maximum likelihood and maximum parsimony was conducted using the D2-D3 sequence data. Of 558 characters, 254 characters (45.5%) were variable and 198 characters (35.4%) were parsimony informative. All phylogenetic methods produced a similar topology with two distinct clades: One clade consists of all Hoplolaimus species while the other clade consists of the rest of the studied Hoplolaiminae genera. This result suggests that Hoplolaimus is monophyletic. Another clade consisted of Aorolaimus, Helicotylenchus, Rotylenchus, and Scutellonema species. Phylogenetic analysis using the outgroup species Globodera rostocheinsis suggests that Hoplolaiminae is paraphyletic. In this study, the D2-D3 region had levels of DNA sequence divergence sufficient for phylogenetic analysis and delimiting species of Hoplolaiminae.     

Genome Comparison and Phylogenetic Analysis of Orientia tsutsugamushi Strains

Orientia tsutsugamushi (OT) is an obligate intracellular bacterium belonging to the family Rickettsiaceae and is the causative agent of scrub typhus, or Tsutsugamushi disease. The complete genome sequences of two OT strains (Boryong and Ikeda) have recently been determined. In the present study, we performed a fine genome sequence comparison of these strains. Our results indicate that although the core gene set of the family Rickettsiaceae is highly conserved between the two strains, a common set of repetitive sequences have been explosively amplified in both genomes. These amplified repetitive sequences have induced extensive genome shuffling and duplications and deletions of many genes. On the basis of the results of the genome sequence comparison, we selected 11 housekeeping genes and carried out multilocus sequence analysis of OT strains using the nucleotide sequences of these genes. This analysis revealed for the first time the phylogenetic relationships of representative OT strains. Furthermore, the results suggest the presence of an OT lineage with higher potential for virulence, which may explain the clinical and epidemiological differences between ‘classic’ and ‘new’ types of Tsutsugamushi disease in Japan.     

High Prevalence of Haplorchiasis in Nan and Lampang Provinces,Thailand, Proven by Adult Worm Recovery from Suspected Opisthorchiasis Cases

Opisthorchiasis, a risk factor for cholangiocarcinoma in humans, is of public health importance in Thailand. The Annual Surveillance Reports from Nan and Lampang Provinces, Thailand, for the year 2011 showed an opisthorchiasis prevalence of over 70% by recovery of eggs in the feces. This study investigated whether most cases are actually due to minute intestinal flukes (MIF) rather than Opisthorchis viverrini, as the eggs of both can hardly be differentiated by morphology. Fifty and 100 cases from residents in Nan and Lampang, respectively, had stools positive for eggs initially assumed to be those of O. viverrini. Each patient was given praziquantel at 40 mg/kg in a single dose. After 2 hr, 30-45 ml of the purgative magnesium sulfate was given, and stools were collected up to 4 times sequentially. The stools were examined for adult worms by simple sedimentation. It was found that 39 of 50 cases (78.0%) from Nan Province had Haplorchis taichui, with intensities ranging from 5 to 1,250 with an average of 62 worms/case. Taenia saginata (7 cases) and Enterobius vermicularis (1 case) were other helminths recovered as the co-infectants. In Lampang Province, H. taichui was recovered from 69 cases (69.0%). The number of flukes recovered ranged from 1 to 4,277, with an average of 326 worms/case. Four cases had Phaneropsolus bonnei, and 10 T. saginata as the co-infectants. Adult specimens of O. viverrini were not recovered from any stool. Clearly, MIF infection, especially haplorchiasis, is more common in northern Thailand. These findings should encourage the Public Health Office to employ more specific tools than Kato''s method for surveillance of opisthorchiasis in Thailand.     

Corbicula fluminea (Bivalvia: Corbiculidae): a possible second molluscan intermediate host of Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea.

More than 1,500 clams of Corbicula fluminea, the most favorable food source of freshwater bivalves in Korea, were collected from 5 localities to examine cercarial and metacercarial infection with Echinostoma cinetorchis. Although 3 clams infected with suspicious E. cinetorchis metacercariae out of 200 specimens collected at Kangjin, Chollanam-do were detected, no cercarial and metacercarial infections with E. cinetorchis were observed in field-collected Corbicula specimens. In the susceptibility experiments with laboratory-reared clams, those infected with miracidia of E. cinetorchis did not release their cercariae up to 60 days after infection. To confirm the identity of second intermediate host of E. cinetorchis experimentally, a total of 30 clams were exposed to the cercariae from Segmentina hemisphaerula that had been infected with miracidia of E. cinetorchis. The clams were susceptible to cercariae of E. cinetorchis with an infection rate of 93.3%. Metacercariae from clams taken more than 7 days after cercarial exposure were fed to rats (S/D strain), and adult worms of E. cinetorchis, characterized by 37-38 collar spines on the head crown, were recovered from the ileocecal regions. This is the first report of C. fluminea as a possible second intermediate host of E. cinetorchis.