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.

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.

Characterization of YS-27, an axenic Korean strain of Entamoeba histolytica.

Characterization of YS-27, an axenic Entamoeba strain, was performed by three different laboratory methods. Zymodeme analysis using starch gel electrophoresis and PCR with species-specific primers showed that YS-27 is a pathogenic Entamoeba which belongs to the group II zymodeme. Pathogenicity of YS-27 was further confirmed by observing the formation of liver abscess in Mongolian gerbils. These results showed that YS-27 is E. hisolytica.

Cloning of a cysteine proteinase cDNA of adult Paragonimus westermani by polymerase chain reaction.

Cysteine proteinase cDNA fragment from adult mammalian trematode, Paragonimus westermani was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends (RACE). It has an open reading frame of 804 bp. The deduced amino acid sequence consists of 268 amino acids. Sequence analysis and alignment showed significant sequence similarity to other eukaryotic cysteine proteinases and conservation of the cysteine, histidine, and asparagine residues that form the catalytic triad. The cysteine proteinase cDNA fragment was also subcloned in the expression vector pET and expressed as a C-terminal His-tag fusion protein in Escherichia coli.

Biostatic activity of Coix lacryma seed extract on Toxoplasma gondii in macrophages.

Water extract of Coix lacryma seeds (Co-Ex) was separated into several components; dissolved with Tris-Cl buffer and the supernatant (WC1), ammonium sulfate treatment supernatant (WC2) and the pellet (WC3), QAE column chromatography of WC1 and the peak portions; WC4, WC5 and WC6. Murine peritoneal macrophages in DMEM containing 10% heat-inactivated FCS were infected with tachyzoites of Toxoplasma gondii, RH strain, in vitro. By adding modulators such as Co-Ex, WC1,2,3,4,5,6 and LPS or IFN-gamma for 24 hrs, toxoplasmastatic activity of macrophages was examined in relation to nitrite production. Nitrite production of macrophages was enhanced especially in the series of WC2, WC1 and the combination sample (WC1+WC2+WC3) by order, than other components or fractions (WC4, WC5, WC6) tested. Toxoplasmastatic actions such as percentage of the macrophages infected by T. gondii and fold increase of T. gondii in macrophages showed retroverse relations with the amount of nitrite production; i.e., as nitric oxide (NO) increased the phagocytic index of macrophages and the fold increase of tachyzoites in macrophages decreased. Nitrite (NO2) production was increased by adding IFN-gamma in all cases together with enhancement of biostatic effects. Through the results obtained, it is speculated that some components other than the non-proteinous and defatted components in Coix lacryma seeds may contribute to activate macrophages through induction of NO for the biostatic activity.

Axenization of Entamoeba histolytica, a Korean strain YS-27.

In the present communication, YS-27, a Korean strain of Entamoeba histolytica is described for the isolation and establishment of axenic cultivation. E. histolytica, designated as strain "YS-27", was isolated from the pus of a hepatic abscess obtained from a 72 year old inpatient of August 10, 1969. Specimens, were obtained by needle aspiration, inoculated immediately and weekly cultured in a modified diphasic medium at 37 degrees C. Strain YS-27 had been maintained for more than 15 years by weekly subculture until February, 1985. These cultures were transferred to a monoxenic TTY-SB medium seeded with a trypanosomatid of the genus Crithidia. Penicillin G, 2 to 10 x 10(3) International units and Streptomycin, 2 to 10 mg per 100 ml, were added to the cultures to eliminate the bacteria. After more than one year later, these two organisms were well maintained by transfer every 3 or 4 days until January, 1986 at 37 degrees C in TTY-SB medium in the absence of other microorganisms. These monoxenic cultures were then transferred to TYI-S-33 medium. Strain YS-27 alone had not been growing at the time of transfer, but when overlaid with Crithidia at intervals of 3 to 4 days, strain YS-27 propagated well. The Crithidia died out several weeks later after several passages. Beginning in April, 1986, strain YS-27, was successfully established in axenic culture in TYI-S-33 medium and has been maintained in continuous culture and multiplied well to present.

Cytotoxicity of a cysteine proteinase of adult Clonorchis sinensis.

To clarify the correlation of the proteinase activity with pathogenicity of Clonorchis sinensis, the proteinase activity either in excretory-secretory products (ESP) or in crude extracts of adult C. sinensis was examined. Substrate gel electrophoresis of the ESP and crude extracts revealed four distinct enzyme bands, which were differently inhibited by the specific proteinase inhibitors. The proteinase of the ESP with molecular mass of 24 kDa, was purified 23-fold with 14.5% yield by spectra gel ACA 44 gel filtration. It exhibited optimal pH at 7.5 in sodium phosphate (0.1 M). Its activity was inhibited specifically by N-ethylmaleimide (NEM) and antipain whereas potentiated 1.9 folds in the presence of 5 mM dithiothreitol (DTT). Cytotoxicity of the proteinase increased in a dose-dependent manner up to 120 micrograms/ml while reduced by NEM and antipain, indicating that cysteine proteinase was responsible for the cytotoxicity. This result shows that the 24 kDa cysteine proteinase is deeply correlated with the pathogenicity of C. sinensis infection.

Nitric oxide mediates the toxoplasmastatic activity of murine microglial cells in vitro.

Toxoplasma gondii (T. gondii), an opportunistic protozoan, is an important cause of central nervous system (CNS) infections in immunosuppressed patients. The present study focused on the interaction between T. gondii and microglial cells from the brain of neonatal Balb/c mice. Preincubation of the murine microglial cells with recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) induced significant inhibition of T. gondii replication in a dose dependent manner. This antiparasitic effect in microglial cells was correlated with the induction of the L-arginine-dependent generation of reactive nitrogen intermediates (RNIs). Tumor necrosis factor-alpha (TNF-alpha) was also involved in the toxoplasmastatic activity. Microglial cells incubated with recombinant TNF-alpha in combination with a non-activating concentration of rIFN-gamma released substantial amount of RNIs. Neutralizing antibodies against mouse TNF-alpha inhibited the release of RNI by rIFN-gamma activated macrophages. In summary, the present results show that activation of microglial cells by rIFN-gamma and LPS induce the production of nitric oxide (NO) by these cells via an L-arginine dependent pathway. NO appears to be the effector molecule mediating the toxoplasmastatic effects in these cells.

[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.

Eating habits of east Asian people and transmission of taeniasis.

In order to understand the role of raw meat and viscera eating habits in the transmission of taeniasis in Asian countries, 1502 infected aborigines in ten mountainous districts/towns of six counties in Taiwan, 58 infected persons in two villages on Cheju Island, Korea, and 97 cases in Ambarita District on Samosir Island, North Sumatra, Indonesia were studied during the field surveys. All infected Taiwan aborigines had the habit of eating raw meat and viscera of wild and/or domestic animals. Among these aborigines, 73% ate wild boar, 66% flying squirrel, 65% wild goat, 56% muntjac, 49% wild rats, 46% monkey, 38% hare, 20% civet-cats, 18% weasel, 17% pheasant, 14% squirrel, 4% grouse, 1% deer, 1% snake, less than 1% bamboo partridge, less than 1% frog, less than 1% bear, less than 1% dog, and less than 1% fox. Of the 58 infected persons with Taenia on Cheju Island, Korea, 72% ate raw meat and/or viscera of pig and cattle, 19% raw pork only, and 9% raw beef only. Among 12 infected persons infected with T. saginata-like tapeworms, 7 had eaten raw pork, 2 raw beef and pork and 3 raw pork. Almost all of the 97 cases of taeniasis on Samosir Island of North Sumatra, Indonesia, had eaten only undercooked pork. Eleven of 15 cases were found to be infected with T. saginata-like tapeworms. Eating habits observed suggest an unusual way of transmission of Taenia in East Asia.

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.

Current status of food-borne parasitic zoonoses in Korea.

In Korea meat from various mammals, reptiles, amphibians, fish, and crustaceans serves as host for approximately 25 species of parasites. These food-borne parasitic zoonoses are important public health problems; they are of concern for the live stock and food industry and for farmers and fisherman. Traditionally, Koreans have been vegetable and fish eating people, but eating habits are changing to include the eating of meat. Dogs, cats and other pets are finding places in the home and these animals become involved in zoonotic diseases. Consequently, the prevalence of zoonotic parasitoses is increasing in Korea. There are increasing reports of cryptosporidiosis and trichinosis in the country associated with changing cultural habits in the Korean people.

[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.

Overview of Entamoeba histolytica and amebiasis in Korea with special reference to research notes.

Amebiasis caused by Entamoeba histolytica infection has been a prevalent protozoan disease in Korea since old times. For this reason, a number of scientific publications in relation to this protozoa has totaled 186 so far in the literature, as of 1989. The subjects ranged from biology to its treatment and prevention. The overall achievement by scientists and clinicians in Korea has been meritorious in elucidating some of the pending problems in the areas of pathogenesis, diagnosis, and treatment. Among them, development of a culture method applying diphasic medium, studies on virulence of the strains collected in Korea, and a combination of aspiration method and chemotherapy to treat amebic liver abscess are seen to have been contributing to the eventual goal of reducing this protozoa infection from the land.

Experimental studies of Korea Taenia (Cheju strain) infection in domestic animals.

In the present study, one DYL pig (five-day old), four L-SEM pigs (37-day old), three SEM pigs (44-day old), one Holstein calf (eight-day old) and two Sannean goats (five-day old) were each fed 1000, 30,000, or 380,000 eggs of the Korea Taenia (Cheju strain) and were sacrificed 16-105 days after inoculation. All DYL, L-SEM, and SEM pigs and the Holstein calf were susceptible to Korea Taenia, and one of the two Sannean goats became infected. The cysticerci recovery rates for L-SEM, SEM and DYL pigs, Holstein calf, and Sannean goat were 5.6, 1.7, 0.06, 0.03 and 0.02% respectively. Cysticerci were recovered only from the livers of the infected animals, and more cysticerci were found in the parenchyma (51%) than on the surface of the liver (49%). Immature cysticerci were first observed on day 16 after infection, mature ones on day 29, and degenerated or calcified cysticerci on day 30. Inoculation of a huge number (380,000) of Korea Taenia eggs lead to early degeneration or calcification of the cysticerci. The length, the width, and the diameters of protoscolex, rostellum, and sucker of the cysticerci were largely proportional to the period of inoculation. The measurements and patterns of the hooklets indicate that Korea Taenia is very similar to Taiwan Taenia but is different from T. saginata and T. solium. The results of the present study provide evidence that Korea Taenia and Taiwan Taenia may be of the same species.

Clonorchis sinensis: experimental and clinical studies with praziquantel in Korea.

EXPERIMENTAL STUDIES: A high degree of prophylactic effect was achieved with a single dose of praziquantel (2-cyclohexylcarbonyl-1,2,3,6,7,11b-hexahydro-4H-pyrazino [2,1-a]isoquinolin-4-one, EMBAY 8440, Biltricide), 600 mg/kg bwt once a day, when given to rats at least one day before the infection. To treat the larvae or immature worms at early stages of infection, the following regimens were recommended: 2 X 50 mg/kg bwt for 5 days (Soh et al. 1980) or 3 X 100 mg/kg bwt for 2 days. By morphological examination, it was found that praziquantel induced chronic degeneration of all organs of the parasite such as vacuolization of secretory granules in the integument, degenerative changes of mitochondria and so on. The vacuoles might fuse with each other, and eventually form a balloon-like structure on the tegumental layer causing disruption. CLINICAL STUDIES: Among various regimens with different dosages, administration of 3 X 25 mg/kg bwt for 2 days showed the best results with complete cure. 53 cases were treated, 33 receiving a dosage of 3 X 25 mg/kg bwt on a single day and 20 receiving the same dosage on two days; the former achieved 99.4% egg reduction rate and 96.9% complete cure, and the latter dosage resulted in 100% complete cure. Side effects such as epigastric pain, nausea, lassitude and headache were complained of in 4 cases among the 53, but were mild and transient. Clinico-physiological examinations during treatment were within normal limits. Clinical symptoms disappeared within one month after treatment. A similar result was reported by other authors, by one of them in one heavily infected case (EPG 452,633).

Single-day drug treatment of amebic liver abscess.

This double-blind trial was initiated to test, in a controlled study, the value of single-day drug treatment of amebic liver abscess. Patients were treated with 2 g either of ornidazole or tinidazole, two newly developed 5-nitroimidazole derivatives, given in two equal doses during the course of 1 day. A success rate of over 94% in the 72 patients from both treatment groups who were followed up for 6 months demonstrates the efficacy of the single-day drug treatment.

Transplacental migration of Toxocara canis larvae in experimentally infected mice.

In experimentally infected, nonpregnant mice the larvae of Toxocara canis were not found in the uterus at any time. In mice infected at 1 week, but not at 2 weeks, before gestation, larvae were found in the uterus but not in either the placenta or fetus. In mice infected during pregnancy, larvae were found in the uterus and placenta from the 9th day and in the fetus from the 11th day of pregnancy, more abundantly when infected at the middle than at the earlier stages. Examination of microsections revealed larvae in both maternal sinusoidal spaces and fetal blood vessels of the placenta; though mechanical damage to the tissues and the debris of tissues were sometimes seen, larva-associated inflammation in these tissues were not observed. The results suggest that in the pregnant mouse the migration of T. canis larvae is influenced by the developmental stages of the placenta.