Host specificity of Pisidium coreanum (Bivalvia: Sphaeriidae) to larval infection with a human intestinal fluke Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea.

The fingernail clam, Pisidium coreanum, has been traditionally consumed raw as a so-called drug therapy by patients with bone fractures in Korea. The present study was designed to determine the possible occurrence and, if present, the prevalence of Echinostoma cinetorchis in P. coreanum collected at a local site, and to determine the susceptibility of the clams in the laboratory to infection with miracidia and cercariae of E. cinetorchis. No cercariae or metacercariae of E. cinetorchis were observed in field-collected P. coreanum clams. In susceptibility experiments with laboratory-reared clams, individuals exposed to miracidia of E. cinetorchis did not release cercariae by 20 days after exposure; necropsy of exposed clams failed to show development of any sporocysts or rediae. To confirm the possibility of these clams serving as an experimental second intermediate host of E. cinetorchis, 20 of them were exposed to E. cinetorchis cercariae from experimentally infected Segmentina hemisphaerula that had been previously exposed to miracidia of E. cinetorchis; all exposed clams became infected. Metacercariae from clams at 14 days postinfection were fed to rats, and adult worms were recovered from the ileocecal regions. This is the first report of P. coreanum serving as second intermediate host of E. cinetorchis.

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.

Segmentina hemisphaerula: a new molluscan intermediate host for Echinostoma cinetorchis in Korea.

Three species of Planorbidae have been reported from Korea, e.g., Gyraulus convexiusculus, Hippeutis (Helicorbis) cantori, and Segmentina (Polypylis) hemisphaerula. Of these, only H. cantori was reported as the first and second intermediate host for Echinostoma cinetorchis, an important human intestinal parasite in Korea. Segmentina hemisphaerula has also been found to be an intermediate host. In field-collected planorbids, only S. hemisphaerula was found shedding echinostome cercariae and infected with metacercariae of E. cinetorchis, whereas no G. convexiusculus and H. cantori were found to be infected. In experiments with laboratory-bred snails, G. convexiusculus and S. hemisphaerula were susceptible to infection by miracidia of E. cinetorchis, but H. cantori could not be infected. Tadpoles of Rana nigromaculata and laboratory-bred snails of the 3 planorbid species were exposed to E. cinetorchis cercariae shed from field-collected S. hemisphaerula. All tadpoles, S. hemisphaerula, and G. convexiusculus became infected, but no H. cantori were infected. Metacercariae from tadpoles, S. hemisphaerula, and G. convexiusculus were fed to rats per os, and eggs of E. cinetorchis were detected in the rat feces 1 wk later. The rats were killed, and adult E. cinetorchis were recovered from the small intestines. This is the first report of G. convexiusculus as a potential first and second intermediate host and of S. hemisphaerula as a new first and second intermediate host for E. cinetorchis in Korea.

Austropeplea ollula (Pulmonata: Lymnaeidae): a new molluscan intermediate host of a human intestinal fluke, Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea.

Three freshwater snail species of the family Lymnaeidae have been reported from Korea, Radix auricularia coreana, Austropeplea ollula and Fossaria truncatula. Out of 3 lymnaeid snail species, A. ollula was naturally infected with the Echinostoma cinetorchis cercariae (infection rate = 0.7%). In the experiments with the laboratory-bred snails, F. truncatula as well as A. ollula was also susceptible to the E. cinetorchis miracidia with infection rates of 25% and 40%, respectively. All of three lymnaeid snail species exposed to the E. cinetorchis cercariae were infected with the E. cinetorchis metacercariae. It is evident that A. ollula acts as the first molluscan intermediate host of E. cinetorchis in Korea, and F. truncatula may be a possible candidate for the first intermediate host of this intestinal fluke. Also, three lymnaeid snail species targeted were experimentally infected with E. cinetorchis metacercariae.

Immunoelectron microscopic localization of partially purified antigens in adult Paragonimus iloktsuenesis.

An immunoelectron microscopy employing immunogold labeling method was performed to detect tissue origin of D1 fraction (D1A) among 5 antigenic protein fractions partially purified by DEAE-anion exchange chromatography from water-soluble crude antigen (PIWA) of adult Paragonimus iloktsuenensis. Immune reactions of adult worm tissues with rabbit serum immunoglobulin immunized with crude antigen (PI-Ig) and D1 antigen (D1-Ig), as well as rat serum immunoglobulin infected with P. iloktsuenensis were observed. D1A showed strong antigenicity in the intestinal epithelium of the worms during the early infection period of 2-4 weeks after infection. The vitellaria also showed stronger antigenicity than the other tissue sites in immune reaction of tissues against all immunoglobulins from 4 to 33 weeks after vitelline development. Therefore, it is suggested that D1A was mainly originated from the intestinal epithelial tissues before the development of vitelline gland of the parasites. Immuno-reactivity of two immunoglobulins (PI-Ig, D1-Ig) was significantly different in intestinal epithelial cytoplasmic protrusions (CP) and intestinal epithelial secretory granules (SG). In the experimental group with D1-Ig, gold particles were labeled significantly in CP than in SG when compared to the PI-Ig group. Thus, the major antigenic materials in D1 antigen having a strong antigenicity in the early infection period was considered to be originated from the intestinal epithelial tissue.

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.

Non-specific activation of mouse peritoneal macrophages by a freshwater ciliate, Tetrahymena pyriformis.

Toxoplasma-killing activities of mouse peritoneal macrophages activated by the extracts of Tetrahymena pyriformis (Korean and Chinese strains) were evaluated, and the active protein fractions from both strains were partially characterized by a method including chromatographies and SDS-PAGE. The first peak in Korean strain and the second peak in Chinese strain of T. pyriformis obtained by DEAE-Sephadex A-50 chromatography were most effective in the activation of macrophages to kill Toxoplasma gondii tachyzoites in vitro. Subsequent fractionations of obtained peak fractions were performed on a Sephadex G-200 gel. The first peaks fractionated from both strains of T. pyriformis had the highest toxoplasmacidal activities, and when subjected to the SDS-PAGE, one prominent band was visualized for each of the strains showing the same molecular weight of ca. 52.6 kDa. This active protein is suggested to be related to non-specific activation of mouse peritoneal macrophages.

Molecular cloning and expression of Cu/Zn-containing superoxide dismutase from Fasciola hepatica.

The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.

Cipangopaludina chinensis malleata (Gastropoda: Viviparidae): a new second molluscan intermediate host of a human intestinal fluke Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea.

Two species of the viviparid snails have been reported in Korea, e.g., Cipangopaludina chinensis malleata and Cipangopaludina japonica. Cipangopaludina chinensis malleata collected at 3 of 12 localities were found to be infected with metacercariae of Echinostoma cinetorchis, one of the snail-borne human intestinal trematodes in Korea. Metacercariae from these snails 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. However, no C. japonica collected from 2 localities harbored the metacercariae. In experiments with laboratory-bred viviparid snails, all viviparids were not susceptible to miracidia of E. cinetorchis. To confirm the identity of second intermediate hosts of E. cinetorchis experimentally, 2 species of viviparid snails were exposed to the cercariae from Segmentina hemisphaerula that had been infected with miracidia of E. cinetorchis. Both species of snails were susceptible to cercariae of E. cinetorchis. This is the first report of Cipangopaludina spp. serving as the second intermediate host of E. cinetorchis and as a potential source of human infection.

Karyotypes on three species of Chinese mesogastropod snails, Semisulcospira libertina, S. dolichostoma and Viviparus rivularis.

Three species of the families Viviparidae and Pleuroceridae, the first intermediate host of paragonimiasis, metagonimiasis and echinostomiasis were studied cytologically. The observed diploid chromosome number was as follows: Semisulcospira libertina 36, S. dolichostoma 34, and Viviparus rivularis 64. The mitotic chromosome complement of S. libertina has nine metacentric pairs and nine submetacentric pairs, and S. dolichostoma has three metacentric pairs and 14 submetacentric pairs of chromosomes. Viviparus rivularis showed two metacentric pairs and 30 submetacentric pairs of chromosomes.

Planorbid snails as potential molluscan intermediate host of a human intestinal fluke, Neodiplostomum seoulensis (Trematoda : Diplostomatidae) in Korea.

Three species of the pulmonate snails of the family Planorbidae have been reported from Korea; Gyraulus convexiusculus, Hippeutis (Helicorbis) cantori and Segmentina (Polypylis) hemisphaerula. Of these 3 species, only H. cantori is reported as the molluscan intermediate host of Neodiplostomum seoulensis, one of the important snail-borne human intestinal trematodes in Korea. However, S. hemisphaerula was also found to be an intermediate host for N. seoulensis. In field-collected snails, H. cantori and S. hemisphaerula were found shedding bifurcated cercariae of N. seoulensis, whereas no G. convexiusculus was found shedding cercariae. In experiments with laboratory-bred snails, only S. hemisphaerula was susceptible to miracidia of N. seoulensis. Tadpoles of Rana nigromaculata and R. rugosa were exposed to cercariae shed from field-collected and laboratory-bred S. hemisphaerula. All tadpoles of R. nigromaculata were found to be massively infected, but none of the tadpoles of R. rugosa were infected with larvae of N. seoulensis. Metacercariae from tadpoles of R. nigromaculata and the snake Rhabdophis tigrinus tigrinus were fed to rats, and eggs of N. seoulensis were detected in the rat feces one-week later. These rats were killed and adult N. seoulensis recovered from the small intestines. This is the first report of S. hemisphaerula as a molluscan intermediate host for N. seoulensis in Korea.

[Five human cases of Diphyllobothrium latum infection through eating raw flesh of redlip mullet, Liza haematocheila].

A total of 32 human diphyllobothriasis cases have been reported so far in Korea, excluding 21 egg-positive cases from stool examinations. Authors experienced five more human cases of Diphyllobothrium latum infection, especially infected due to eating raw flesh of redlip mullet, Liza haematocheila. Five cases were neighbors residing in the Puchon area, Kyonggi-do, who ate raw mullets (L. haematocheila) in a party in February 1996. The mullets were purchased at the Noryangjin fisheries market in Seoul. All of cases (2 males and 3 females) were 35 to 43 years old and healthy with the body weight range of 56-62 kg. They complained about gastrointestinal trouble and abdominal discomfort, but were in normal ranges of their hematology and urinalysis data. None revealed any sign of anemia. The patients experienced natural discharge of a chain of segments before, and showed diphyllobothriid eggs in their stool specimens when they visited our laboratory. They were administered with praziquantel (15 mg/kg of body weight) and 30 g of magnesium sulfate as a purgative. Two whole worms with the scolices (310-340 cm in length; 8-13 mm in width) were expelled each from two out of five cases after anthelmintic treatment, and the others expelled the parts of strobilae without scolex. The worms were identified as D. latum, based on the following biological characters: external morphologies, coiling of uterus, the number of uterine loops, position of genital opening, morphologies of cirrus, cirrus sac and seminal vesicle on the histological sections, position of vagina and uterine pore, and microscopical and SEM morphologies of the eggs.

[A human case of internal myiasis in Korea].

A 71-year old male patient was admitted in Inha hospital due to right facial palsy, quadriplegia and aphasia. This patient was operated to replace his heart values 7 years ago and has been treated with Coumarin, an anti-coagulant drug, to prevent the formation of thrombus in the heart. A number of fly maggots continuously crawled out from the nasogastric tube set up for supplying a liquid diet and patient's mouth for 2 days until his death in the intensive care unit of hospital. These maggots were about 11.5 mm long on the average and identified as genus Lucilia belonging to family Calliphoridae. The lesion of this myiasis case might be regarded in the gastro-intestinal system of patient. This is the first report of an internal myiasis case in Korea.

Segmentina (Polypylis) hemisphaerula (Gastropoda: Planorbidae): a new molluscan intermediate host of a human intestinal fluke Neodiplostomum seoulensis (Trematoda: Diplostomatidae) in Korea.

Three species of the pulmonate snails of the family Planorbidae have been reported from Korea, Gyraulus convexiusculus, Hippeutis (Helicorbis) cantori, and Segmentina (Polypylis) hemisphaerula. Of these 3 species, only H. cantori is reported as the molluscan intermediate host of Neodiplostomum seoulensis, 1 of the important snail-borne human intestinal trematodes in Korea. However, S. hemisphaerula was also found to be an intermediate host for N. seoulensis. In field-collected snails, H. cantori and S. hemisphaerula were found shedding bifurcated cercariae of N. seoulensis, whereas no G. convexiusculus was found shedding cercariae. In experiments with laboratory-bred snails, only S. hemisphaerula was susceptible to miracidia of N. seoulensis. Tadpoles of Rana nigromaculata and Rana rugosa were exposed to cercariae shed from field-collected and laboratory-bred S. hemisphaerula. All tadpoles of R. nigromaculata were found to be massively infected, but none of the tadpoles of R. rugosa was infected with larvae of N. seoulensis. Metacercariae from tadpoles of R. nigromaculata and the snake Rhabdophis tigrinus tigrinus were fed to rats, and eggs of N. seoulensis were detected in the rat feces 1 wk later. These rats were killed and adult N. seoulensis recovered from the small intestines. This is the first report of S. hemisphaerula as a molluscan intermediate host for N. seoulensis in Korea.

[Proteinase activity in the isolates of Trichomonas vaginalis according to their pathogenicity].

Ten axenic isolates of Trichomonas vaginalis were subcutaneously injected to the BALB/c mice in order to assess their pathogenicity by means of so-called "mouse assay" method. All the isolates revealed neutral and acid proteinase activities both in their lysates and in culture media, but the specific activities of both proteinases in the severely pathogenic group were significantly higher than the mildly pathogenic group (p < 0.05). In the SDS-PAGE system in which the electrophoretic gels contained 0.4% gelatin as the substrate, five different banding patterns of trichomonal proteinases were detected, and the patterns were closely related with the pathogenicity of the isolates of T. vaginalis. All five bands might be regarded as cysteine proteinases group in the inhibitor assays. The cytotoxicity of the lysates of T. vaginalis to the target Chinese hamster ovarian (CHO) cell line was also significantly different according to the pathogenicity of the isolates, and generally lower in the lysates treated with cysteine proteinase inhibitors than in the control lysates. In summarizing the results, it might be considered that the proteinases of T. vaginalis showing five electrophoretic banding patterns are closely related with the pathogenicity and cytotoxicity of the isolates of T. vaginalis.

[Enterobius gregorii Hugot, 1983 recovered from school children in Kangwon-Do, Korea].

Hugot (1983), Hugot and Tourte-Schaefer (1985) reported that nematodes of the genus Enterobius causing human enterobiasis might be classified into two different species on the basis of the size and shape of spicule in male worms. E. gregorii Hugot, 1983 has significantly shorter spicules in males than those in E. vermicularis. A total of 194 male Enterobius were collected from 17 schoolchildren by anthelmintics at Wonju, Kangwon-do. The sizes of spicules and morphologies were comparatively observed. Two different types of spicules in male Enterobius were also observed in Korea. Out of 112 male worms observed, the percentages of E. gregorii (short-spicule type) and E. vermicularis (long-spicule type) were 23.2% and 76.8%, respectively.

Diagnosis of clonorchiasis by ELISA-inhibition test using a Clonorchis sinensis specific monoclonal antibody.

The ELISA-inhibition test using Clonorchis sinensis specific monoclonal antibody (CsHyb 0605-23) for diagnosis of clonorchiasis was carried out. It demonstrated sensitivity and high specificity in comparison with the conventional ELISA.

Snail-borne parasitic zoonoses in Korea.

A total of 22 snail-borne parasites causing various parasitic zoonoses in Korea are listed and reviewed. All of these parasites are indigenous except Heterophyes heterophyes, H. dispar and Angiostrongylus cantonensis detected in patients who traveled outside of Korea.

Analysis of Clonorchis sinensis antigens and diagnosis of clonorchiasis using monoclonal antibodies.

Clonorchis sinensis is a common parasite of man in Korea. Researches on the specific antigens of C. sinensis would be valuable not only because those elucidate the molecular characteristics of this fluke but also because it is applicable to immunodiagnosis. Although many monoclonal antibodies have been used in the field of parasite immunology, few articles on monoclonal antibodies against C. sinensis have been published so far. The aim of this study was to analyze C. sinensis antigens recognized by monoclonal antibodies, and to set up ELISA-inhibition test using C. sinensis specific monoclonal antibodies for improved specificity of immunodiagnostic tests. By fusion between spleen cells of the mice immunized with C. sinensis water-soluble crude adult worm antigens and plasmacytoma cells of mouse origin, 29 hybridoma clones secreting anti-C. sinensis monoclonal antibodies were made, and 8 clones among those were found specific. After cell cloning, isotypes of 6 selected specific monoclonal antibodies were determined to be IgG1, IgG2b and IgA. Four exposed antigenic determinants of natural infection were recognized by different specific monoclonal antibodies. By enzyme-immunoelectrotransfer blot, 10 KD, 34 KD antigenic determinants were found to be reacted with CsHyb 0714-20, CsHyb 0605-10 monoclonal antibodies, respectively. The antigenic determinant recognized by CsHyb 0714-20 monoclonal antibody was revealed to be located at the surface and parenchyme of a parasite by indirect immunofluorescent antibody technique, and those reacted with CsHyb 0605-10, CsHyb 0714-25 monoclonal antibodies were found at the parenchyme and intestine. The antigenic determinant reacted with CsHyb 0605-23 monoclonal antibody was found mainly around the uterine eggs. Four antigenic determinants recognized by specific monoclonal antibodies were all found to be present in the early eluted fractions of C. sinensis antigens separated by Sephadex G-200 gel filtration. By conventional ELISA, 75% of clonorchiasis cases were found positive, but 7.1% of normal controls and 37.5% of paragonimiasis cases showed false positives. However, by ELISA-inhibition test using C. sinensis specific monoclonal antibody (CsHyb 0605-23), 77.1% of clonorchiasis cases were found positive, and there were no false positives in normal controls or paragonimiasis cases, indicating 100% specificity.(ABSTRACT TRUNCATED AT 400 WORDS)

[Effect of in vivo administration of Tetrahymena pyriformis on the in vitro toxoplasmacidal activity of mouse peritoneal macrophages].

Tetrahymena pyriformis is a free-living ciliate protozoan in the freshwater system. Experiments were carried out to determine whether intraperitoneal administration of T. pyriformis (GL strain) to mice activates macrophages to be able to kill Toxoplasma gondii tachyzoites in vitro. Mice were also injected intraperitoneally with several synthetic activators; dimethyldioctadecylammonium bromide (DDA), dextran sulfate, complete Freund's adjuvant (CFA) as well as Toxoplasma and Tetrahymena lysates in order to activate mouse peritoneal macrophages. One week after the administration of activators, peritoneal cells were harvested and the adherent macrophages were challenged with Toxoplasma tachyzoites. Macrophage monolayers were then fixed with absolute methanol after washing, and stained with Giemsa solution. The percentage of the adherent cells infected and total number of organisms per 100 macrophages were calculated to make toxoplasmacidal activity of macrophages according to the cultivation time. Peritoneal macrophages from mice administered with Tetrahymena exhibited significant protection against target parasites as compared with those treated with synthetic activators. Among non-biological synthetic activators, DDA was evaluated as an excellent activator.