Heterophyid metacercarial infections in brackish water fishes from Jinju-man (Bay), Kyongsangnam-do, Korea

Heterophyid metacercarial infections in brackish water fishes, i.e., perch, shad, mullet, redlip mullet, and goby, of Jinju-man (Bay), Kyongsangnam-do, Korea, were investigated using a digestion technique. Among 45 perch (Lateolabrax japonicus), the metacercariae of Heterophyopsis continua were found in 55.6% (18.5 metacercariae per fish), Stictodora spp. in 28.9% (3.6), and Metagonimus takahashii in 6.7% (17.0). The metacercariae of H. continua were detected in 23 (65.7%) of 35 shad (Konosirus punctatus). Among 15 mullet (Mugil cephalus), the metacercariae of Pygidiopsis summa were found in 100% (105.9 metacercariae per fish), Heterophyes nocens in 40.0% (8.5), H. continua in 13.3%, and Stictodora spp. in 6.7%. Among 12 redlip mullet (Chelon haematocheilus), the metacercariae of P. summa were detected in 91.7% (1,299 metacercariae per fish), H. nocens in 16.7%, and Stictodora spp. in 16.7%. Among 35 gobies (Acanthogobius flavimanus), the metacercariae of Stictodora spp. were found in 82.9% (44.5 metacercariae per fish), and H. continua in 25.7% (2.8). The adult flukes recovered from a cat experimentally infected with metacercariae from perches were compatible with M. takahashii. The results confirm that the estuarine fish in Jinju-man (Bay) are heavily infected with heterophyid metacercariae. Furthermore, it was found for the first time that the perch acts as a second intermediate host for M. takahashii.     

Echinostoma hortense and Heterophyid Metacercariae Encysted in Yellowfin Goby, Acanthogobius flavimanus, from Shinan-gun and Muan-gun (Jeollanam-do), Korea

Fishborne trematode (FBT) metacercariae were investigated in yellowfin goby, Acanthogobius flavimanus, collected from Shinan-gun and Muan-gun, Jeollanam-do (province), Korea. All collected fishes were examined using the artificial digestion method. In all of 15 gobies from Aphae-myeon in Shinan-gun, metacercariae of Stictodora spp. (334 metacercariae/fish), Heterophyes nocens (153/fish), and Heterophyopsis continua (20/fish) were detected. In 2 of 14 gobies from Jido-myeon in Shinan-gun, 8 Echinostoma hortense metacercariae in total were detected. In 15 gobies from Haeje-myeon in Muan-gun, the metacercariae of H. continua were found in 100%, Stictodora spp. in 86.7%, and H. nocens in 6.7% of fish examined. The average numbers of metacercariae per infected fish were 23.3 (H. continua), 416.0 (Stictodora spp.), and 2.0 (H. nocens), respectively. The metacercariae of E. hortense found in gobies were elliptical, with 150 × 138 µm in average size, and had 27 collar spines on the head crown. The above results suggest that yellowfin gobies from 2 localities may be the potential infection sources of FBT. Moreover, it is proved for the first time that the yellowfin goby, A. flavimanus, acts as a second intermediate host for E. hortense.     

A survey of Heterophyes nocens and Pygidiopsis summa metacercariae in mullets and gobies along the coastal areas of the Republic of Korea

The infection status of mullets Mugil cephalus (n = 139) and gobies (n = 35) Acanthogobius flavimanus with metacercariae of Heterophyes nocens and Pygidiopsis summa was examined in 11 western, southern, and eastern coastal areas of the Republic of Korea, using a digestion technique. Heterophyid metacercariae were highly prevalent in mullets from western and southern coastal areas; Shinan-gun (100% for H. nocens and 100% for P. summa), Muan-gun (93% and 100%), Buan-gun (42% and 75%), Seocheon-gun (73% and 53%), Ganghwa-gun (47% and 100%), Sacheon-shi (47% and 77%), and Gangjin-gun (50% and 70%, respectively). Only 1 (10%) of 10 mullets from an eastern coastal area, i.e., Donghae-shi, was positive for P. summa metacercariae. Metacercarial densities were the highest in the trunk of mullets for H. nocens and the gill for P. summa. Gobies from Muan-gun were positive for H. nocens (40%) and P. summa metacercariae (40%), and gobies from Seocheon-gun revealed H. nocens metacercariae (20%). The metacercarial density was remarkably higher in mullets than in gobies. The results revealed that H. nocens and P. summa metacercariae are prevalent in mullets and gobies from coastal areas of the Republic of Korea, and the prevalence and intensity of infection vary according to geographical locality.     

Antibody-dependent rat macrophage-mediated damage to the excysted metacercariae of Paragonimus westermani in vitro

An in vitro immune effector mechanism against the target excysted metacercariae of Paragonimus westermani was demonstrated in the rat system. Peritoneal exudate cells, mainly macrophages from normal rats, showed adherence to and killing of excysted metacercariae of P. westermani in the presence of complement-independent serum from rats infected with Paragonimus metacercariae. These reactions were specific for the excysted metacercariae, as tissue-migrating juvenile worms were not affected. Damage of excysted metacercariae of P. westermani due to antibody and macrophages was assessed by morphological observation, by cell adherence reaction and by the use of vital dyes. Trypan blue dye exclusion proved to be a reliable indicator of judging metacercarial viability. Electron microscopic studies demonstrated that macrophages reacted with fuzzy material on the tegumental surface and fine structures in the syncytium of the parasites. The tubular tunnels formed between the basement membrane and muscle layers of the damaged parasites were also noticeable. The relevance of these findings to cellular immunity in the early paragonimiasis was discussed.     

Clonorchis sinensis metacercarial infection in the pond smelt Hypomesus olidus and the minnow Zacco platypus collected from the Soyang and Daechung Lakes

The pond smelt Hypomesus olidus and minnow Zacco platypus were collected from the Soyang and Daechung Lakes in January 2003, and their metacercarial infections was examined by the muscle compression and artificial digestion techniques. In the Soyang Lake, 161 metacercariae of Clonorchis sinensis (0.35 per fish) were harvested from 459 pond smelts examined. Also, 13 metacercariae of C. sinensis (0.43 per fish), 1 of Metagonimus sp., 4 of Echinostoma sp., 148 of Centrocestus armatus and 44 unidentified species were collected from 30 minnows. In the Daechung Lake, 369 metacercariae of C. sinensis (3.69 per fish) and 51 unidentified species were recovered from 100 pond smelts. The metacercariae of C. sinensis were fed to experimental rats, in which the adult flukes were identified. The pond smelts and minnows collected from the Soyang and Daechung Lakes were verified to be the second intermediate hosts and the sources of human C. sinensis infection.     

Scanning electron microscopy and chemical excystation of Fascioloides magna (Trematoda) metacercariae

Scanning electron microscopy was used to study encysted metacercariae and newly excysted juveniles of Fascioloides magna. The outer cyst was rough, coarse and discontinuous in the ventral aspect; the inner cyst was smooth. The newly excysted metacercaria was plump and contained numerous tegumentary spines; large dome-shaped papillae were prominent around the oral sucker and on the rim of the acetabulum. Encysted metacercariae with outer cysts were excysted in an alkaline bile salts-trypsin medium at an elevated temperature in the absence of acid saline or acid pepsin pretreatment. Pretreatment in acid saline slightly decreased subsequent excystation, while pretreatment in acid pepsin slightly enhanced subsequent excystation in the alkaline bile salts-trypsin medium.     

Histochemical and thin layer chromatographic analyses of neutral lipids in Echinostoma revolutum metacercariae cultured in vitro

Excysted metacercariae of Echinostoma revolutum cultured in vitro in the defined medium, NCTC 135 supplemented with 20% hens' egg yolk, doubled their mean relative body area and showed significant sucker growth within 14 days. Histochemical Oil Red O staining showed neutral fat mainly in the excretory system of excysted metacercariae and in adults grown in the domestic chick. In vitro cultured worms showed neutral fat in the intestine, parenchyma, and excretory system. As detected by TLC the major neutral lipid fractions were free fatty acids for excysted metacercariae; free sterols for adults grown in chicks; and triglycerides, free fatty acids, and free sterols for cultured worms. Excysted metacercariae excreted free fatty acids into a nonnutrient incubation medium, whereas cultured worms excreted diglycerides, triglycerides, and free fatty acids into the medium.     

Effect of cobalt-60 irradiation on the infectivity of Paragonimus westermani metacercariae.

A study was made to observe the effect of cobalt-60 irradiation on the viability of Paragonimus westermani metacercariae in Sinopotamon chekiangense crabs. The crabs were collected in mountain regions of the Zhejiang Province of China in which paragonimiasis is endemic. Adult cats and albino mice were infected with metacercariae irradiated at different doses. Dissection of the host animals was conducted 90 or 30 days, respectively, after infection for recovery of lung flukes. Anti-metacercariae antibody in infected mice was measured by enzyme-linked immunosorbent assay (ELISA). Results showed that metacercariae were unable to grow into adult worms in cats after exposure to gamma irradiation at a dose of 0.10 kGray. However, a small number of metacercariae exposed to a dose of 2.0 kGray excysted and survived in 1 mouse for 30 days. No worm was recovered from mice when the metacercariae were irradiated at a dose of 2.5 kGray. Seropositive results by ELISA were obtained when the mice were infected with metacercariae irradiated at doses ranging from 2.0 to 3.5 kGray.     

Identification of Stictodora lari (Heterophyidae) metacercariae encysted in the brackish water fish, Acanthogobius flavimanus

Metacercariae of the genus Stictodora encysted in the head tissue of Acanthogobius flavimanus (the gobies) caught at Sachun-gun, Kyongnam Province, were identified to be Stictodora lari Yamaguti, 1939 (Trematoda: Heterophyidae), a new parasite fauna in Korea. The metacercariae were 0.39-0.43 mm by 0.32-0.35 mm in size, long elliptical, and with a thin and transparent cyst wall. Total 200 metacercariae were collected from 50 gobies. In order to obtain adult worms two kittens and a puppy were infected each with 34-100 metacercariae, and total 33 adults were recovered between the day 4 and day 8 post-infection. The S. lari adults measured 0.95-1.18 mm long and 0.26-0.32 mm wide and the eggs in uteri 0.028-0.033 mm by 0.017-0.020 mm. The most characteristic morphological feature of these flukes was the presence of a gonotyl and gonotyl spines arranged in two groups; densely crowded group of 30-40 spines and linearly-arranged one of 30-40 spines, together of which made a comma (or reversed comma) shape along the lateral margin of the gonotyl. It has been proved by this study that S. lari is distributed in southern coasts of Korea.     

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy,morphometry and confocal laser scanning microscopy

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy.     

Gynaecotyla squatarolae (Digenea: Microphallidae) from rats experimentally infected with metacercariae from the shore crab, Macrophthalmus dilatatus

Metacercariae of Gynaecotyla squatarolae (Digenea: Microphallidae) were discovered from the shore crab, Macrophthalmus dilatatus, purchased at a market in a coastal town of Taean-Eup, Chungcheongnam-do, Republic of Korea. Their adult flukes were confirmed by experimental infection of rats. The metacercariae of G. squatarolae were elliptical (391.1 x 362.5 micrometer), and the excysted metacercariae had progenetic genital organs, including the ovary and testes. To obtain adult flukes, 6 Sprague-Dawley rats were fed 500 metacercariae each, and killed at days 2, 4, and 6 post-infection. The adult flukes were identified as G. squatarolae (Yamaguti, 1934) Yamaguti, 1939, based on morphological characters, including 2 ventral suckers (1 large and 1 small), a large genital atrium equipped with the cirrus and the metraterm, separated male and female genital pores, a transversely long cirrus pouch, and extensive vitelline follicles. In the present study, it has been first proven that the shore crab M. dilatatus is a second intermediate host for G. squatarolae in the Republic of Korea.     

Infection Status of Estuarine Fish and Oysters with Intestinal Fluke Metacercariae in Muan-gun, Jeollanam-do, Korea

The source of human infection with intestinal flukes was surveyed in estuarine fishes, including the dotted gizzard shad, common sea bass, common blackish goby, redlip mullet, black sea bream, and oyster collected from Muan-gun, Jeollanam-do, Korea during August and September 2007. Collected fishes and oysters were artificially digested in pepsin-HCl solution and examined under a stereomicroscope. In 36 shads (Konosirus punctatus) and 20 basses (Lateolabrax japonicus) examined, Heterophyopsis continua metacercariae were found in 58.3% and 100%, and their average numbers were 12.0 and 6.3 per infected fish, respectively. In 34 gobies (Acanthogobius flavimanus) examined, metacercariae of H. continua were detected in 79.4%, Stictodora lari in 97.1%, and Acanthotrema felis in 92.1%, and their average numbers were 45.8, 189.3, and 235.3 per infected fish, respectively. In 37 redlip mullets (Chelon haematocheilus), Heterophyes nocens metacercariae were found in 56.8%, Pygidiopsis summa in 94.6%, and Stictodora fuscata in 45.9%, and the average metacercarial densities were 17.4, 31.3, and 35.1 per infected fish, respectively. In 30 black sea breams (Acanthopagrus schlegeli) and 45 oysters (Crassostrea gigas) examined, no metacercariae were detected. From the above results, it has been confirmed that the dotted gizzard shad, common sea bass, common blackish goby, and redlip mullet from Muan-gun, Jeollanam-do, Korea are infected with the metacercariae of heterophyid flukes.     

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.     

Use of an aceto-carmine procedure to examine the excysted metacercariae of Echinostoma caproni and E. trivolvis.

A simple aceto-carmine procedure was developed to relax, fix, stain, and clear excysted metacercariae of Echinostoma caproni and E. trivolvis. This procedure allowed for morphologic details on whole metacercariae comparable to those seen with more elaborate staining procedures. Differences in dimensions and staining intensities between the two species of metacercariae are described.     

Growth and feeding of Echinostoma revolutum on the chick chorioallantois and in the domestic chick

Chemically excysted metacercariae of Echinostoma revolutum cultivated on the chick chorioallantois grew slowly until day 5, more rapidly between 5 and 7 days, and slowly between 7 and 10 days. Worms did not become ovigerous in this site by 12 days, at which time studies were terminated. In contrast, chemically excysted metacercariae reared in the domestic chick were ovigerous by day 9, at which time their mean body area was about 4 times greater than the largest chorioallantoic worms. Histochemical studies, solubility tests for hematin, and X-ray microanalysis of cecal contents showed that chorioallantoic-worms fed on blood from the vascular membrane, whereas chick-worms fed on host intestinal mucosa.     

Studies on host specificity in Paragonimus westermani: II. Histochemical and cytochemical characterization of metacercariae and worms from rats and dogs

Histochemical tests were done on newly excysted metacercariae and worms recovered from an abnormal host (rat) and the definitive host (dog) for some oxidoreductases, phosphatases and glycosidases. The results demonstrate that rat worms have enzymatic distribution and intensities more similar to those of metacercariae than to adult worms from dogs. Ultracytochemical examination of acid and alkaline phosphatase and Mg-ATPase activity was also carried out. Acid phosphatase activity occurred exceptionally in the excretory bladder and caeca of dog worms. No activity was observed in rat worms except for lysosomal granules in the tegument. Alkaline phosphatase activity was exhibited in the excretory bladder in both dog and rat worms. Mg-ATPase activity occurred in the tegument and parenchymal cells in dog worms and in the excretory bladder in rat worms. In metacercariae, little or no reaction for these enzymes was present except for Mg-ATPase activity on the excretory ducts. These observations, together with the histochemical results, indicate that metabolic activity in rat worms is higher than in metacercariae although it is strongly reduced compared with dog worms.     

In vivo and in vitro studies of the effects of immune rat serum on Fasciola hepatica

Significant protection against infection with 10 or 30 metacercariae of Fasciola hepatica was conferred on naive rats by the passive transfer of serum derived from rats which had been exposed to primary and challenge infections with 5 or 10 and 30 or 20 metacercariae respectively. Immune serum did not have a pronounced effect on the mortality of metacercariae in vitro. However, its presence was associated with the formation of a precipitate on the tegument of each metacercaria and in the culture medium. The precipitate contained rat antibody and other components, presumably parasite antigens, which elicited the formation of antibody when the precipitate was injected into rats. Viability of metacercariae cultured in immune and normal sera as well as freshly excysted specimens was tested in rats by intraperitoneal infection. Metacercariae cultured in immune serum did not develop. By comparison with the viability of freshly excysted metacercariae, that of some metacercariae cultured in normal serum was impaired; this was attributed to inadequacies in the culture technique. A relationship between precipitate formation in vitro and impaired viability of metacercariae in vivo has yet to be established.     

Excretory-secretory product of newly excysted metacercariae of Paragonimus westermani directly induces eosinophil apoptosis

Eosinophils are important effector cells in host defense against parasites. Excretory-secretory product (ESP) produced by helminthic worms plays important roles in the uptake of nutrients, migration in the host tissue, and in immune modulation. However, little is known about the ability of the ESP to directly trigger eosinophil apoptosis. This study investigated whether the ESP of newly excysted metacercariae of Paragonimus westermani could induce apoptosis in human eosinophils. Apoptosis was assayed by staining the cells with FITC-annexin V, and the cells were analyzed by flow cytometry. It was found that the ESP of newly excysted metacercariae of P. westermani induced a direct time- and concentration-dependent increase in the rate of constitutive apoptosis in mature human eosinophils. Eosinophil apoptosis was first apparent 3 hr after treatment with the ESP and continued to increase after 6 hr of incubation with respect to the cells cultured in the absence of the ESP. While only 2.8% of the eosinophils incubated in the medium for 3 hr were apoptotic, 7.6%, 10.9% and 22.6% of the eosinophils treated with 10, 30 and 100 micrograms/ml ESP were apoptotic, respectively. This result suggests that the ESP of newly excysted metacercariae of P. westermani directly induce eosinophil apoptosis, which may be important for the survival of the parasites and the reduction of eosinophilic inflammation in vivo.     

Identification of Acanthotrema felis (Digenea: Heterophyidae) metacercariae encysted in the brackish water fish Acanthogobius flavimanus

The metacercariae of Acanthotrema felis Sohn et al., 2003 (Digenea: Heterophyidae) were discovered in a species of the brackish water fish, Acanthogobius flavimanus, in the Republic of Korea. They were experimentally fed to kittens, and adult flukes were harvested 7 days later. The adults were morphologically characterized by the presence of a bipartite seminal vesicle, the ventral sucker associated with a ventrogenital sac enclosing 3 sclerites (2 long and pointed, and 1 short and thumb-like), and an unarmed gonotyl. The adult flukes were identified as A. felis Sohn et al., 2003, and the brackish water fish A. flavimanus has been verified as one of its second intermediate hosts.