A new neodiplostomid (Digenea) from the intestine of chicks infected with metacercariae from the grass snake, Rhabdophis tigrina

Three species of neodiplostomula are known to inhabit the European grass snake, Rhabdophis tigrina, in the Republic of Korea: Pharyngostomum cordatum (large-sized neodiplostomula), an intestinal trematode of cats; Neodiplostomum seoulense (small-sized neodiplostomula), an intestinal trematode of humans and rodents; and Neodiplostomum leei (small-sized neodiplostomula), which migrates to the livers of rodents and is an intestinal trematode of birds. The present study describes a fourth species, Neodiplostomum (Conodiplostomum) boryongense n. sp. (Digenea: Neodiplostomidae), based on adult flukes recovered from the small intestines of chicks experimentally infected with small-sized neodiplostomula from the grass snake. The new species differs from 13 previously known species. It also differs from N. seoulense in its larger body size, severely bilobed testes, and smaller genital atrium, and from N. leei in its larger body size, smaller ventral sucker, presence of a genital cone, and vitelline follicles distributed chiefly in the forebody. The new species does not migrate to the livers of rodents nor does it develop to adulthood in the rodent intestines. However, the neodiplostomula of the new species are indistinguishable from those of the other 2 species. Results show that at least 4 species of neodiplostomula inhabit the grass snake in the Republic of Korea.     

Migration of Neodiplostomum leei (Digenea: Neodiplostomidae) neodiplostomula to the livers of various mammals

Of morphologically indistinguishable small-sized neodiplostomula in the grass snake Rhabdophis tigrina in the Republic of Korea, some, such as Neodiplostomum seoulense, mature into adults in the intestines of rodents, whereas others, such as Neodiplostomum leei, migrate to rodent livers and mature in the intestines of chicks. In the present study, we aimed to observe in more detail the extraintestinal migration and development of N. leei by using various animal models, i.e., mice, rats, hamsters, rabbits, cats, and chicks. In mammals, small-sized neodiplostomula (N. leei) inoculated orally penetrated the intestinal wall, entered the peritoneal cavity, and oriented to the liver without passing through any other organ. In rodent livers, the neodiplostomula were surrounded by host inflammatory cells and fibrotic tissues from day 10 postinfection (PI); the worms were found dead by day 56 PI. When neodiplostomula from rodent livers were transferred orally to mammals, they reoriented to the liver, although they were able to develop into adults in the chick intestine by day 6-7 PI. The possibility of human infections, i.e., liver migration, by N. leei neodiplostomula, among snake consumers, warrants investigation.     

Antigenic properties of cystatin-binding cysteine proteinases from Neodiplostomum seoulense

The antigenic properties of cysteine proteinases binding to cystatin were analyzed in Neodiplostomum seoulense, an intestinal trematode that infects humans and rodents in the Republic of Korea. Cystatin was found to effectively capture cysteine proteinases present in the crude extract of N. seoulense. The IgG levels against cystatin-binding cysteine proteinases in sera of mice infected with N. seoulense were higher than those in sera of mice immunized with the crude extract of N. seoulense. The production of IgG antibodies against cystatin-binding cysteine proteinases increased according to the length of infection period. In immunoblots of purified cystatin-binding proteinases, 2 molecules, approximately 50 kDa and 60 kDa, reacted with N. seoulense-infected mouse sera. Of the sera from patients infected with various helminths, those of sparganum-infected patients showed the strongest affinities for cystatin-binding cysteine proteinases of N. seoulense. Cystatin-binding cysteine proteinases of N. seoulense are suggested to be putative antigens for serodiagnosis of human N. seoulense infection.     

Metacercariae of Pharyngostomum cordatum found from the European grass snake, Rhabdophis tigrina, and its experimental infection to cats

The metacercariae of Pharyngostomum cordatum were found naturally infected in the European grass snake, Rhabdophis tigrina, purchased from a local snake collector in Jinju, Kyongsangnam-do. They were experimentally fed to several kinds of animals such as mice, rats, hamsters, ducklings, a dog, and cats. The adult worms were recovered from the cats 5 weeks after the infection, but none from other animals. The measurements and other morphological characters of the metacercariae and adults were both compatible with those of P. cordatum described by previous authors. The present study confirmed that the snake, Rhabdophis tigrina, serves as a second intermediate (or paratenic) host of P. cordatum in Korea.     

Microphallus koreana n. sp. (Trematoda: Microphallidae) transmitted by a marine crab, Macrophthalmus dilatatus

Microphallus species occur primarily as intestinal parasites of birds and mammals, and metacercariae of a new species belonging to this genus have been discovered from the crab, Macrophthalmus dilatatus, in the Republic of Korea. The metacercaria of this fluke was round with 2 thick walls, and the excysted one had mature genital organs. The adult flukes recovered from experimentally infected chicks had numerous intrauterine eggs, well-developed pars prostatica, widely bifurcating ceca, and prominent uterine bulge. After observing internal structures, it was concluded that this species is different from any other known Microphallus spp. Based on the morphology of metacercariae and adult flukes, we describe this specimen as a new species, Microphallus koreana n. sp.     

Food-borne intestinal trematode infections in the Republic of Korea

A total of 19 species of food-borne intestinal trematodes have been reported in humans in the Republic of Korea. They include 12 species of the Heterophyidae, Metagonimus yokogawai, M. takahashii, M. miyatai, Heterophyes nocens, H. heterophyes (imported), H. dispar (imported), Heterophyopsis continua, Pygidiopsis summa, Stellantchasmus falcatus, Centrocestus armatus, Stictodora fuscata, and S. lari; four species of the Echinostomatidae, Echinostoma hortense, E. cinetorchis, Echinochasmus japonicus, and Acanthoparyphium tyosenense; and one species each of the Neodiplostomidae, Neodiplostomum seoulense, Plagiorchiidae, Plagiorchis muris, and Gymnophallidae, Gymnophalloides seoi. Fresh water fish harbor the metacercarial stage of M. yokogawai, M. takahashii, M. miyatai, C. armatus, E. hortense, E. cinetorchis, E. japonicus, or P. muris. Brackish water fish serve as the second intermediate hosts for H. nocens, H. continua, P. summa, S. falcatus, S. fuscata, and S. lari. Brackish water bivalves are the source of infection with A. tyosenense. Tadpoles and frogs are the second intermediate hosts for N. seoulense, but the major source of human infection is the grass snake Rhabdophis tigrina, a paratenic host. The metacercariae of G. seoi are observed in oysters. The natural definitive hosts are, in most cases, mammals such as rats, cats and dogs. However, several species (C. armatus, S. lari, E. japonicus, A. tyosenense, and G. seoi) have birds as natural definitive hosts. Host-parasite relationships, pathogenesis and pathology, immunity, clinical aspects, differential diagnosis, treatment, prevention and control of these intestinal trematodes are briefly discussed.     

Apodemus agrarius as a new definitive host for Neodiplostomum seoulense

A total of 1,496 rodents and insectivores were live-trapped at Yeoncheon-gun (n = 351), Paju-shi (804), and Pocheon-gun (343), Gyeonggi-do (Province), and examined for intestinal helminths, including Neodiplostomum seoulense, seasonally from December 2004 to September 2005. Six species of rodents, including Apodemus agrarius (1,366), Mus musculus (32), Micronytus fortis (28), Eothenomys regulus (9), Micronys minutus (6), and Cricetulus triton (3), and 1 species of insectivores Crocidura lasiura (54) were collected. A total of 321 adult N. seoulense were collected from 19 (1.4%) A. agrarius. The worm burden ranged from 1 to 101 per A. agrarius (mean; 16.9). No N. seoulense was observed in other rodent or insectivore species examined. The infection rate during autumn (4.5%) was higher than those during spring (0.8%), summer (0.8%), and winter (0.5%). The average number of N. seoulense in infected A. agrarius was the highest in spring (66.0 specimens), followed by autumn (15.2), winter (4.5), and summer (3.3). This study first confirms that A. agrarius is a natural definitive host for N. seoulense, and demonstrates that the infection rates and intensities vary seasonally and geographically.     

Trematodes Recovered in the Small Intestine of Stray Cats in the Republic of Korea

In 2005, we reported the infection status of 438 stray cats with various species of intestinal helminths, including nematodes (4 species), trematodes (23 species), and cestodes (5 species) in the Republic of Korea. However, morphologic details of each helminth species have not been provided. In the present study, we intended to describe morphologic details of 13 trematode species which were either new fauna of cats (10 species) or new fauna of not only cats but also all animal hosts (3 species). The worms were fixed in 10% neutral buffered formalin under a cover slip pressure, stained with Semichon''s acetocarmine, and then observed using a light microscope equipped with a micrometer. The 13 subjected species included members of the Heterophyidae (Stellantchasmus falcatus, Stictodora fuscata, Stictodora lari, Centrocestus armatus, Procerovum varium, and Cryptocotyle concava), Echinostomatidae (Echinostoma hortense, Echinostoma revolutum, Echinochasmus japonicus, and Stephanoprora sp.), Diplostomidae (Neodiplostomum seoulense), Plagiorchiidae (Plagiorchis muris), and Dicrocoeliidae (Eurytrema pancreaticum). By the present study, Cryptocotyle sp. and Neodiplostomum sp. recored in our previous study were identified as C. concava and N. seoulense, respectively. Three species, P. varium, C. concava, and Stephanoprora sp., are new trematode fauna in Korea.     

Phylogenetic Relationships of 3 Korean Neodiplostomum Species (Digenea: Neodiplostomidae) Based on Partial CO1 Gene

The phylogenetic relationships of the 3 Neodiplostomum spp. (Digenea: Neodiplostomidae) occurring in Korea (N. seoulense, N. leei, and N. boryongense) were analyzed using the partial mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. The adult flukes were recovered from Sprague-Dawley rats (N. seoulense) and newborn chicks (N. leei and N. boryongense) experimentally infected with the neodiplostomula from the grass snake, Rhabdophis tigrinus tigrinus. The genomic DNA was amplified using specific primers, and the sequence of CO1 was obtained. According to the results, the pairwise similarity was 96.1% between N. boryongense and N. seoulense, but was 95.0% between N. boryongense and N. leei and 94.2% between N. leei and N. seoulense. The results demonstrated a closer phylogenetic relationship between N. seoulense and N. boryongense. This high relationship of N. seoulense and N. boryongense may be related to their similar morphologic features including the limited distribution of vitellaria and the presence of a genital cone. N. leei is distinct on the other hand with an extensive distribution of vitellaria and the absence of a genital cone.     

Infection status with helminthes in feral cats purchased from a market in Busan, Republic of Korea

The present study was performed to investigate the infection status with helminth in a group of feral cats in Korea. More than 29 helminth species including adults or eggs were detected in visceral and fecal samples of the examined cats. Among these were a host of nematodes, including toxocarids, Ancylostoma sp. and the larva of Anisakis simplex; trematodes, including Clonorchis sinensis, Paragonimus westermani, Eurytrema pancreaticum, Pharyngostomum cordatum, Metagonimus spp., Heterophyes nocens, Pygidiopsis summa, Heterophyopsis continua, Stictodora fuscata, Stictodora lari, Acanthotrema felis, Stellantchasmus falcatus, Centrocestus armatus, Procerovum varium, Cryptocotyle sp., Echinostoma revolutum, Echinostoma hortense, Echinochasmus japonicus, Stephanoprora sp., Plagiorchis muris, Neodiplostomum sp. and diplostomulum. We also detected a variety of cestodes, including Spirometra erinacei, Taenia taeniaeformis and unidentified species of tapeworm. We also found examples of the acanthocephalan, Bolbosoma sp. In our assessment of the stools, we detected at least 12 species of helminth eggs. These findings confirmed that feral cats in Korea are infected with a variety of helminth parasite species. Furthermore, among the helminths detected, E. pancreaticum, S. fuscata, S. lari, A. felis, S. falcatus, C. armatus, P. varium, Cryptocotyle sp., E. revolutum, E. japonicus, Stephanoprora sp., P. muris, Neodiplostomum sp. and Bolbosoma sp. represent helminth fauna which have not been reported previously in feral cats in the Republic of Korea.     

Host specificity of Austropeplea ollula (Gastropoda: Lymnaeidae) to miracidial infection with a human intestinal fluke Neodiplostomum seoulense (Trematoda: Diplostomatidae) in Korea

Three species of freshwater pulmonate snails of Lymnaeidae have been reported from Korea, Radix auricularia coreana, Austropeplea ollula, and Fossaria truncatula. Of these 3 species, only A. ollula was naturally infected with Neodiplostomum seoulense cercariae (prevalence, 0.3%). In experiments with the laboratory-reared snails, A. ollula was found to be susceptible to the N. seoulense miracidia (prevalence, 10%). This is the first report of A. ollula as the first molluscan intermediate host for N. seoulense in Korea.     

Depressed neuronal growth associated protein (GAP)-43 expression in the small intestines of mice experimentally infected with Neodiplostomum seoulense

Neodiplostomum seoulense (Digenea: Neodiplostomidae) is an intestinal trematode that can cause severe mucosal pathology in the small intestines of mice and even mortality of the infected mice within 28 days after infection. We observed neuronal growth associated protein-43 (GAP-43) expression in the myenteric plexus of the small intestinal wall of N. seoulense-infected mice until day 35 post-infection (PI). BALB/c mice were infected with 200 or 500 N. seoulense metacercariae isolated from naturally infected snakes and were killed every 7 days for immunohistochemical demonstration of GAP-43 in the small intestines. N. seoulense-infected mice showed remarkable dilatation of intestinal loops compared with control mice through days 7-28 PI. Conversely, GAP-43 expression in the mucosal myenteric plexus was markedly (P<0.05) reduced in the small intestines of N. seoulense-infected mice during days 7-28 PI and was slightly normalized at day 35 PI. From this study, it is evident that neuronal damage occurs in the intestinal mucosa of N. seoulense-infected mice. However, the correlation between intestinal pathology, including the loop dilatation, and depressed GAP-43 expression remains to be elucidated.     

Discovery of Maritrema jebuensis n. sp. (Digenea: Microphallidae) from the Asian Shore Crab, Hemigrapsus sanguineus, in Korea

Maritrema spp. (Digenea: Microphallidae) are parasites of birds, but have not been found in the Republic of Korea. In this study, metacercariae of Maritrema sp. were discovered in the Asian shore crab, Hemigrapsus sanguineus, caught in the mud-flats of Jebu-do, Hwasung-gun, Gyeonggi-do, and the adult flukes were confirmed by experimental infection into mice. Based on the symmetric ribbon-like vitellarium, adult flukes of Maritrema sp. were identified, but did not belong to previously described species in terms of the following morphologic characteristics: ceca reaching to the lateral wall at the anterior border of the ovary; ventral sucker larger than oral sucker; a prominent metraterm; and vitellarium forming a complete ring. Hence, we named this microphallid M. jebuensis n. sp. after the island where the second intermediate hosts were collected. From this study, it has been shown that Maritrema sp. is distributed in Korea and transmitted by the Asian shore crab, H. sanguineus.     

Icosiella turgeocauda n. sp. (Nematoda: Onchocercidae) and Seuratascaris numidica (Nematoda: Ascarididae), parasites of the frog,Rana cancrivora (Anura: Ranidae), from Luzon,Republic of the Philippines

Icosiella turgeocauda n. sp. from the intestinal mesenteries of Rana cancrivora collected at Luzon, Republic of the Philippines, is described and illustrated. Icosiella turgeocauda n. sp. represents the ninth species to be assigned to the genus and is easily differentiated from all the previously described species by the position of the vulva and the presence of bilateral umbos on the caudal end of the male. Seuratascaris numidica also was found. The Philippines represents a new location record for S. numidica.     

A new gymnophallid trematode from the intestine of mice infected with metacercariae from the razor clam Sinonovacula constricta

Meiogymnophallus sinonovaculae n. sp. (Digenea: Gymnophallidae) is described from metacercariae found in the razor clam, Sinonovacula constricta, in the Republic of Korea, and adults recovered from the small intestine of experimentally infected mice. The worms are characterized by paired clusters of vitelline follicles, a well-developed pars prostatica with abundant prostate cells, a voluminous undivided seminal vesicle (in adults and metacercariae), and large, grouped, domelike sensory papillae on the ventral surface anterior to the ventral sucker (in metacercariae). This new species resembles the type species Meiogymnophallus affinis, but differs in having compact, elliptical, and 4-5-lobed vitellaria, and an excretory vesicle with bicornuated anterior arms reaching to the oral sucker. This is the second documentation of the presence of a species of Meiogymnophallus in the Republic of Korea.     

Stephanostomum lophii sp. nov. (Digenea: Acanthocolpidae), intestinal parasite of Lophius piscatorius, with reference to seasonal fluctuations of metacercariae in intermediate second hosts (Gadidae)

We describe the adult and metacercaria of a new species of digenetic trematode, Stephanostomum lophii sp. nov., an intestinal parasite of the angler fish, Lophius piscatorius . The species is distinguished from the similar Stephanostomum kovalevi by the numerous peristomial spines in the latter and from S. baccatum by the oral sucker being larger than the ventral sucker and by the fact that the principal intermediate hosts are gadids, not pleuronectids as in the case of Stephanostomum baccatum . We also report the prevalence of S. lophii metacercariae in various potential angler fish prey species caught off the coast of Galicia (NW Spain).     

Heydenius dominicus n. sp. (Nematoda: Mermithidae), a Fossil Parasite from the Dominican Republic

Heydenius dominicus n. sp. is described as a new species of fossil mermithid nematode from Dominican Republic amber. The species is represented by two specimens of parasitic juveniles that left their insect host and became embedded in the resin. The nematodes are associated with an adult male limoniid (Diptera: Limoniidae) and an adult female mosquito (Diptera: Culicidae). The parasites are thought to have emerged from the mosquito host. This is the first report of a fossil mermithid from a Neotropical area.     

Acanthoparyphium shinanense n. sp. (Digenea: Echinostomatidae) from Experimental Chicks Infected with Metacercariae Encysted in Brackish Water Clams in the Republic of Korea

Acanthoparyphium shinanense n. sp. (Digenea: Echinostomatidae) is described from chicks experimentally infected with the metacercariae encysted in 2 brackish water clam species, Ruditapes philippinarum and Coecella chinensis, in the Republic of Korea. The metacercariae were round to oval, armed with 23 collar spines, and 0.216 (0.203–0.226) mm in diameter. From 5 chicks experimentally infected each with 200 metacercariae, 34 juvenile (5-day-old worms) and 104 adult flukes (7-day-old worms) were harvested from their small intestines, with the average worm recovery rate of 13.8%. The adult flukes were 3.18 (2.89–3.55) mm long and 0.68 (0.61–0.85) mm wide, with an elongated, posteriorly tapering body, and a prominent head collar armed with 23 collar spines arranged in a single uninterrupted row. The posterior testis of A. shinanense was longitudinally elongated, which is similar to Acanthoparyphium spinulosum Johnston, 1917 but unique from the other closely related species, including Acanthoparyphium tyosenense Yamaguti, 1939, Acanthoparyphium kurogamo Yamaguti, 1939, and Acanthoparyphium marilae Yamaguti, 1934. The eggs of A. shinanense were larger than those of A. spinulosum, and the anterior extent of 2 lateral groups of vitellaria was slightly more limited in A. shinanense than in A. spinulosum. Molecular analysis of nuclear and mitochondrial genes revealed low homology with A. spinulosum from USA (96.1% in 5.8S rRNA) and Ukraine (97.9% in 28S rRNA), Acanthoparyphium n. sp. from USA (98.0% in 28S rRNA), and Acanthoparyphium sp. from Australia, Kuwait, and New Zealand. Biological characteristics, including its first intermediate host and natural definitive hosts, as well as its zoonotic capability, should be elucidated.     

A Microsporidan Hyperparasite of Strigeoid Trematodes, Nosema strigeoideae sp. n.

SYNOPSIS. Nosema sp. Cort, Hussey & Ameel, 1960, previously reported as a hyperparasite of strigeoid trematode larvae and apparently specific for strigeoids, is described as a new species, N. strigeoideae , with fresh spores averaging 4.7 by 3.1 μ. Primary infection appears to be in the wall of the trematode sporocyst, spreading from there to the developing embryos.