Double-stranded RNA virus in Korean isolate IH-2 of Trichomonas vaginalis.

In this study, we describe Korean isolates of Trichomonas vaginalis infected with double-stranded (ds) RNA virus (TVV). One T. vaginalis isolate infected with TVV IH-2 evidenced weak pathogenicity in the mouse assay coupled with the persistent presence of a dsRNA, thereby indicating a hypovirulence effect of dsRNA in T. vaginalis. Cloning and sequence analysis results revealed that the genomic dsRNA of TVV IH-2 was 4,647 bp in length and evidenced a sequence identity of 80% with the previously-described TVV 1-1 and 1-5, but only a 42% identity with TVV 2-1 and 3 isolates. It harbored 2 overlapping open reading frames of the putative capsid protein and dsRNA-dependent RNA polymerase (RdRp). As previously observed in the TVV isolates 1-1 and 1-5, a conserved ribosomal slippage heptamer (CCUUUUU) and its surrounding sequence context within the consensus 14-nt overlap implied the gene expression of a capsid protein-RdRp fusion protein, occurring as the result of a potential ribosomal frameshift event. The phylogenetic analysis of RdRp showed that the Korean TVV IH-2 isolate formed a compact group with TVV 1-1 and 1-5 isolates, which was divergent from TVV 2-1, 3 and other viral isolates classified as members of the Giardiavirus genus.

Encystment and metacercariae development of Echinostoma cinetorchis cercariae in an in vitro culture system.

For some species of 37-collar-spined Echinostoma, their cercariae successfully encyst and develop to metacercariae in vitro. In our study, we cultured Echinostoma cinetorchis cercariae in 12 different media to study the formation of metacercariae. Locke's solution, medium 199, and RPMI 1640 were used as media for culture. RPMI 1640 produced the highest encystment and normal metacercariae development. The osmolality of the media was related to their ability to encyst and develop. The 0.5 X media induced higher encystment and normal metacercaria formation than the 1x media. The addition of fetal bovine serum to RPMI 1640 increased the level of encystment and normal metacercariae development. In the mixture of 0.5 x RPMI 1640 and 10% fetal bovine serum, encystment was highest, at 96.0%, and the development ratio of normal metacercariae was also the highest, at 91.5%, after 48 hr of cultivation. In a viability test, 7 day- and 14 day-cultured metacercariae were successfully matured to adult worms in experimentally infected rats. These results showed that E. cinetorchis cercariae could be cultured to viable metacercariae in an in vitro culture system and that 0.5 x RPMI 1640 plus 10% fetal bovine serum was the most useful medium for cultivation. This culture system can be adapted for additional studies on the E. cinetorchis life cycle, especially to supply large numbers of metacercariae for other studies on this echinostome.

Identification and characterization of a cathepsin L-like cysteine protease from Taenia solium metacestode.

Taenia solium metacestode, a larval pork tapeworm, is a causative agent of neurocysticercosis, one of the most common parasitic diseases in the human central nervous system. In this study, we identified a cDNA encoding for a cathepsin L-like cysteine protease from the T. solium metacestode (TsCL-1) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an approximate molecular weight of 37.6 kDa which containing a typical signal peptide sequence (17 amino acids), a pro-domain (106 amino acids), and a mature domain (216 amino acids). Sequence alignments of TsCL-1 showed low sequence similarity of 27.3-44.6 to cathepsin L-like cysteine proteases from other helminth parasites, but the similarity was increased to 35.9-55.0 when compared to mature domains. The bacterially expressed recombinant protein (rTsCL-1) did not show enzyme activity; however, the rTsCL-1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. It degraded human immunoglobulin G (IgG) and bovine serum albumin (BSA), but not collagen. Western blot analysis of the rTsCL-1 showed antigenicity against the sera from patients with cysticercosis, sparganosis or fascioliasis, but weak or no antigenicity against the sera from patients with paragonimiasis or clonorchiasis.

Acanthoparyphium tyosenense (Digenea: Echinostomatidae): experimental confirmation of the cercaria and its complete life history in Korea.

Cercaria yamagutii Ito, 1957, was found in the marine mesogastropods Lunatia fortuni and Glossaulax didyma from the tidelands of Simpo located at the estuary of the Mankyoung River, which runs to the western coast of Korea. Metacercariae were found in a marine bivalve Mactra veneriformis after being infected with C. yamagutii experimentally. When a sea gull, Larus crassiostris, was fed with the metacercariae collected from the infected M. veneriformis, adult worms were recovered 10 days later. It was confirmed that the parasites collected from L. crassiostris were Acanthoparyphium tyosenense Yamaguti, 1939. From the results of this life cycle study, it was determined that the first intermediate hosts of A. tyosenense are L. fortuni and G. didyma. The second intermediate and final hosts are M. veneriformis and L. crassiostris, respectively. Mactra veneriformis was experimentally infected with C. yamagutii isolated from L. fortuni and G. didyma by maintaining them in a water tank for 30 min at about 20 C. The cercariae entered M. veneriformis through their incurrent siphons. Five hours after infection, the cercariae tails began to separate from the bodies, and the cercariae formed cysts. Mature cysts were formed 340 hr (14 days) after infection and identified as the metacercariae of A. tyosenense. The prevalence of A. tyosenense metacercariae was 99.5% in naturally infected M. veneriformis. This is the first report of C. yamagutii as the cercaria of A. tyosenense, and the complete life cycle of A. tyosenense was established in Korea.

[Studies on the immunodiagnosis of rabbit clonorchiasis II. Immunoaffinity purification of whole worm antigen and characterization of egg, metacercaria and adult antigens of Clonorchis sinensis]

The sensitivity and specificity of crude and affinity-purified antigens of Clonorchis sinensis obtained from the infected rabbits were studied. Stage-specific antigenic proteins from the eggs, metacercariae and adult worms were characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA) The results were as follows: The antibody-binding antigen (ABA) purified from whole worm crude antigen (WWA) by CNBr-activated Sepharose 4B affinity chromatography made 4 specific bands against rabbit anti-sera on Ouchterlony gel diffusion plate, while WWA made 7 bands. Major WWA protein bands by SDS-PAGE were found at 16,300-18,500 and 28,000-29,000 daltons, while major ABA protein bands were at 18,000-21,000 and 29,000-31,000 daltons. The reactivity of ABA with rabbit anti-sera in ELISA was remarkably less sensitive than that of WWA. Molecular weights of egg antigen (EGA), metacercarial antigen (MEA) and adult worm antigen (WWA) of C. sinensis ranged from 15,000-200,000 daltons, 15,000-100,000 daltons and 11,000-80,000 daltons, respectively. Major WWA proteins consisted mainly of polypeptide bands of low molecular weight, less than 31,000 daltons, while those of EGA and MEA consisted of higher molecular weights than 30,000 daltons. The ELISA reactivities of WWA to rabbit anti-sera were remarkably greater than those of MEA. EGA showed negative reaction throughout the experiments. WWA showed higher optical density (O.D.) than 1.0, when reacted with rabbit anti-sera obtained at 4-6 weeks after the infection. In the rabbit anti-sera later than 12 weeks after the infection, the O.D. reacting with WWA showed a plateau without variation. MEA showed relatively low O.D. values (<0.6), when reacted with anti-sera from lightly infected groups throughout the experiments, although there were some weak positive cases (O.D.>0.6) in heavily infected groups. MEA reacted with rabbit anti-sera showed negative results on Ouchterlony gel diffusion plates. Summarizing the above results, it is suggested that the whole worm antigen prepared from the adult worms of C. sinensis is most highly antigenic. However, this antigen might reveal cross reactions with other trematodes such as Paragonimus westermani, therefore, purification of antigenic proteins from the crude antigen is essential to increase the sensitivity and specificity for the immunodiagnosis of clonorchiasis.

[Chemotherapeutic efficacy of trimethoprim-sulfamethoxazole (Bactrim) in experimental murine toxoplasmosis]

The chemotherapeutic efficacy of trimethoprim-sulfamethoxazole (Bactrim) in mice experimentally infected with Toxoplasma gondii was evaluated. The average survival days and survival rate of mice infected intraperitoneally with 1 x 10(5) trophozoites and treated with Bactrim were compared with those of untreated group. The hematologic findings of blood samples of experimental mice were observed for comparison of side effects between Bactrim and pyrimethamine (Daraprim), the latter of which has been one of the favorable drugs for the treatment of toxoplasmosis. The results are summarized as follows: Bactrim showed a strong evidence of potent anti-Toxoplasma activity. The survival rate of mice administered with 24 mg of Bactrim per mouse per day for 7 days, was 83.3 percent, and the rate was increased to 100 percent in mice administered with two-fold concentrated dose of the drug. The average numbers of white blood cells (W.B.C.) in the mouse groups treated with Bactrim or Daraprim were more increased than those only infected with T. gondi. The mice treated with Daraprim, however, showed remarkably decreased numbers of W.B.C. as compared with those treated with Bactrim. The average numbers of red blood cells (R.B.C.) and platelets both in the drug-treated and untreated T. gondii-infected mice were decreased as compared with normal mice. The numbers of R.B.C. in Daraprim-treated mice, however, were more decreased than in Bactrim-treated mice. The average levels of hemoglobin both in the drug-treated and untreated T. gondii-infected mice were decreased, compared with normal mice. But there was no difference in the levels of hemoglobin between Bactrim- and Daraprim-treated groups. In conclusion, trimethoprim-sulfamethoxazole (Bactrim) was proven to be effective and safe for the treatment of murine toxoplasmosis. The efficacy was comparable with pyrimethamine (Daraprim), but bone marrow depression was less severe with Bactrim treatment.

[Epidemiological survey on Metagonimus yokogawai infection in the eastern coast area of Kangwon-province, Korea]

An epidemiological survey on Metagonimus yokogawai infection was performed in the eastern coast area of Kangwon province. Formalin-ether concentration technique and Stoll's dilution egg counting method were employed to figure out the prevalence and intensity of M. yokogawai infection and eating habits of inhabitants with raw freshwater fish. The results are summarized as follows: The prevalence rate of M. yokogawai infection was 6.6% among a total of 2,357 examinees; 8.6% (115 out of 1,345) in male and 4.0% (40 out of 1,012) in female. The prevalence rates were different by the streams where the examinees were residing: 17.8% along the area of Ohsip-cheon (stream), 11.8% along Maeup-cheon, 1.7% along Hwasang-cheon, 1.3% along Yeongok-cheon, 0.9% along Namdae-cheon, 0.9% along Sa-cheon and 0% in the other streams (Yongchon-cheon, Kangnung Namdae-cheon and Jusoo-cheon) in decreasing order. By social strata, the positive rate was 8.0% (122 out of 1,521) in general inhabitants and 3.9% (33 out of 836) in school children. By the intensity of infection by means of EPG counts in feces; the light infection (less than 400/EPG) was 74.1%, moderate infection (401-1,000/EPG) 17.3% and heavy infection (more than 1,001/EPG) 8.6%. Twelves out of 50 sweetfish (Plecoglossus altivelis) (24%) caught from the streams in eastern coast of Kangwon-Do were infected with metacercariae of M. yokogawai, and the number of larvae detected were 70.7 per fish in average. The metacercarial infection rate in fish were also different by the streams; 100% in Ohsip-cheon of Samcheok-Gun, 14.3% in Sa-cheon, 16.7% in Yeongok-cheon of Myeongju-Gun and 0% in Namdae-cheon of Yangyang-Gun, respectively. Six hundred ninety five (695) out of 1,396 inhabitants and school children (49.8%) were experienced in eating raw freshwater fishes (Plecoglossus altivelis, Tribolodon hakonensis, Coreoperca sp., Moroco sp., Carassius sp. & Cyprinus sp. and Zacco platypus). The data were assayed with the questionnaire collected from the examinees. In summarizing the above results, it is known that the Ohsip-cheon area of Samcheok-Gun is highly infected region, and the other areas of Kangnung Namdae-cheon, Yeongok-cheon and Yangyang Namdae-cheon are very lowly infected or non-infected regions for M. yokogawai infection.

[Echinostoma hortense metacercariae naturally encysted in Odontobutis obscura interrupta (a freshwater fish) and experimental infection to rats]

The metacercariae of an echinostomatid fluke were detected from a freshwater fish, Odontobutis obscura interrupta caught from the Seom river system, Wonseong-gun, Kangwon-do, and experimenally infected into rats to obtain the adult worms for the species identification. A total of 32 metacercariae was detected from 10 fishes (27.8 percent) out of 36 O. obscura interrupta caught from the river. The average size of metacercariae was 153.4 x 149.0 micrometer. Also, it was uniquely specific that thickened membrane was formed around the metacercarial cyst wall. The rats orally infected with those metacercariae were sacrificed 20 days after infection to get the adult worms. The mean length and width of the adult worms were 0.75 cm and 0.13 cm, respectively. The number of collar spines on circumoral disk was 26 to 28, and the end group spines at lateral sides of the oral sucker were 4 on each side. The echinostomatid flukes observed in this study were all identified as Echinostoma hortense according to the morphologies of the cirrus sac, ovary, vitellaria and testes of the adult worms and the shape of eggs. On the other hand, O. obscura interrupta is one of the fresh-water fish that are preferably eaten raw by the rural inhabitants. Although no human case of E. hortense infection by eating O. obscura interrupta has been experienced yet, the possibility is strongly suggested in this study.

[Electron-microscopic studies on fine structure and enzyme activity in the axenic and conventional strains of Entamoeba histolytica]

The metabolism of Entamoeba histolytica would be affected by various environmental factors, and alteration of the environment was known to affect the fine structure of E. histolytica. The present study was designed electronmicroscopically to investigate the ultrastructure and enzyme activities in the axenic and conventional strains of E. histolytica. The trophozoites of axenically cultivated HK-9 strain and conventional YS-27 and YS-49 strains of E. histolytica were collected and fixed with 4 percent paraformaldehyde/0.1 M cacodylate buffer (pH 7.4). After washing them by centrifugation, 1 percent warm agar was added in the sediment. Solidified agar with the trophozoites was cut into 1 mm(3) cubes, and incubated in the various substrates to observe enzyme activities. Then, the specimen was post-fixed with 3 percent glutaraldehyde/0.1 M cacodylate buffer (pH 7.4) and 1 percent osmium tetroxide/0.1 M cacodylate buffer (pH 7.4), dehydrated in ascending ethanol series and embedded in epoxy resin. These were sectioned on an ultramicrotome and observed with a transmission electron microscope. The procedures for the observation of the fine structure were same as the above, except for the incubation in the substrate. The sections were stained with uranyl scetate and lead citrate. For the observation of the surface of the amoebae, scanning electron microscopy was carried out. The results obtained in the present study are summarized as follows: 1. The fuzzy coat around double-layered plasma membrane of E. histolytica was more irregularly and densely distributed in the conventional strains (YS-27, YS-49 strains) than in the axenic strain (HK-9 strain). 2. The endosomes, button bodies and chromatin material were surrounded by a double-layered nuclear membrane having scattered nuclear pores. The paranuclear body, mono- or double-layered vacuoles, vacuolar membrane whorls, rosette-like cylindrical bodies, aggregation of cylindrical bodies and helical bodies were found in the cytoplasm of the amoebae. Helical bodies and glycogen granules were generally abundant, while a few smooth endoplasmic reticula were observed in the cytoplasm. 3. Alkaline phosphatase activity was mainly demonstrated in the plasma membrane, limiting membranes of vacuoles and smooth endoplasmic reticula. ATPase activity was observed in the nucleus, limiting membranes of vacuoles and vacuolar membrane whorls. 4. Acid phosphatase activity was commonly demonstrated in the limiting membranes an contents of vacuoles, lysosome-like organelles, plasma membrane and the button bodies in the nucleus. The activity was more weakly demonstrated in the HK-9 strain than in the other conventional strains of E. histolytica. No peroxidase activity was observed in the amoeba strains employed in the present study. 5. With a scanning electron microscope, no distinct structural differences were observed between the amoeba strains. All the trophozoite forms of the amoebae showed crater-like depressions and rugged features on the outer surface.

[Rhabditis sp. infected cases in rural school children]

Five cases infected with Rhabditis sp. were detected in a survey to examine the stool specimen from rural primary school children. A large number of the larvae of Rhabditis sp. detected by the direct cellophane thick smear were cultured by the filter paper method. The examination was carried out in April through June 1980 in Tangjeong-Myon, Ahsan-Gun, and in September 1983 in Sandong-Eup, Yeongwol-Gun. The results obtained in this study were as follows: Prevalence: Out of 925 children, 5(0.54%) children were found to be positive. The number of the detected larvae was 110/0.1 gm of feces in case 1, 35 in case 2, 130 in case 3, 86 in case 4 and 62 in case 5. Larvae: The larvae were prepared by means of the direct fecal smear and measured by a micrometer equipped in the microscope. Twelve(12) through 15-day old larvae in culture were 197.1 micrometer long in average, and the maximum size of the matured stage larvae was 884.0 x 25.9 micrometer. However, the length variation was ranged as 173.0 to 884.0 micrometer. Adults: The size of clubbed adult female was 1,357 micrometer(1,176~1,419) in length and 80 micrometer(79~82) in width. Length of buccal cavity was 33 micrometer. A long cylindrical esophagus (273 micrometer) of the worms with a valved posterior cardiac bulb and with median bulbar swelling was morphologically indicated. Distance from mouth to vulva was occupied 58 percent of body length. Male worm was 1,006 micrometer (890~1,148) in length and 49 micrometer(48~49) wide. Caudal alae of bursa and spicules (75 micrometer in length) were well developed. Eggs: The oval shaped eggs in the female uterus, when cultured, were 66 x 56 micrometer in size, and the eggs laid by the adult could not be detected. So, reproduction might be through to be ovoviviparity. The five cases were re-examined during the period from the 1st to the 3rd week after stool examinations, but Rhabditis sp. were detected again in 4 cases in 1st week. When they were examined in 3rd week, larvae could not be detected. So, it was thought that the infection of Rhabditis sp. to humans was facultative.

[Distribution Of Medically Important Freshwater Snails And Larval Trematodes From Parafossarulus Manchouricus And Semisulcospira Libertina Around The Jinyang Lake In Kyongsang-Nam-Do, Korea]

The Jinyang Lake is dammed up Nam River, and surrounded by Jinju city and four counties; Jinyang, Sanchong, Hadong, and Sachon in Kyongsang-Nam-Do, Korea. The area around this man-made lake have been known as an endemic focus of clonorchiasis in Korea. The present study was first aimed to know the distribution of freshwater mollusks including medically important snails, and larval trematodes shed from Parafossarulus manchouricus and Semisulcospira libertina. In addtion to above studies, water analyses in each snail habitat were carried out in order to figure out a part of their environmental factors. This malaco-ecological survey was done at the six areas around upper, middle and lower parts of the lake for 4 months, August-November, 1983. Total nine species of freshwater mollusks were collected throughout the study: 4 species of gastropods; Semisulcospira libertina, Cipangopaludina chinensis, Parafossarulus manchouricus and Radix auricularia, and 5 species of bivalves; Unio douglasiae, Anodonta woodiana, Lamprotula gottschei, Corbicula fluminea and Limnoperma lucustris. Out of nine species of freshwater mollusks, three species of gastropods; S. libertina, P. manchouricus and R. auricularia were medically important in terms of the transmission of digenetic trematodes to humans. P. manchouricus and R. auricularia were mainly collected from the shallow ponds and the irrigation channels with the muddy basin, but S. libertina and the bivalves were only collected from the stream of Nam river where the gravels and rocks were dominant. The levels of dissolved oxygen(D.O.) and biochemical oxygen demand (B.O.D.(5)) of the water specimens sampled from the study areas ranged from 6.0 to 9.6 ppm and from 0.4 to 1.6 ppm respectively. As a result, it is considered that water system around the Jinyang Lake might be relatively clean without any heavy pollution of aquatic microorganisms and organic materials during the period of this study. On the other hand, eight metalic constituents from the water samples were also assayed, and all metalic ions detected were remarkably low below the legal criteria. However, calcium ion in the water samples from the habitats of P. manchouricus was considerably higher than others. Infection rates of digenetic trematodes in the snails were 6.9 % in P. manchouricus and 4.8 poercent in S. libertina, respectively. P. manchouricus snails harboring with the cercariae of Clonorchis sinensis were only 0.14 % among the snails examined and other trematode cercariae except cercaria of C. sinensis were; furcocercus cercariae, cercaria of Loxogenes liberum type I and II. S. libertina snails parasitized with the cercariae of Metagonimus yokogawai were 1.5 % out of the snails examined and no cercaria of Paragonimus westermani was found in S. libertina snails in the present study. Digenetic trematode cercariae other then M. yokogawai in S. libertina snails were: Cercaria yoshidae (B type), Cercaria cristata, Cercaria innominatum, Cercaria of Centrocestus formosanus and Cercaria nipponensis.

[Studies On The Inducing Possibility Of Human Visceral Larva Migrans Associated With Eating Habit Of Raw Liver Of Domestic Animals]

To observe the possibility of human visceral larva migrans due to eating of raw liver of domestic animals, especially of cattle, and also to serve as a good reference for adequate sanitary measures, the investigation survey was carried out from May 1975 to May 1976. From the subjects of a l,048 inhabitants (male 558, female 490) in five localities including two Provinces and three different cities, food habit was studied by questionnaire mannual. Larvae isolated from liver tissues of cattle, and pig were identified. Experimental observation on the chicken and mice infected with Toxocara canis was undertaken to draw a assumption of possibility inducing human visceral larva migrans. The results obtained from the present study are summarized. 1. A part of Korean people has the habit to eat the livers of cattle, fowl, pig and dog raw. Eating rate of raw beef liver was 37.8% out of l,048 inhabitants, and its rate was higher markedly in male(57.7%) than in female (15. 1%), and the highest rate among the group of 31-40 years old. Eating rate of raw liver of fowl was 5.9%, pig 5.3%, and dog 2.5%. 2. Larva recovery rate from beef liver was 11.8% out of 195 samples and 72.0% of total detected 1arvae were identified as Toxocara(=Neoascaris) vitulorum. From pig liver, larvae of nematoda were found in 6.4% out of 109 samples but no larva was detected from 120 fowl livers. 3. Larvae detected from one-half of tissues and organs of infected chicken with about 2,000 Toxocara canis eggs were 8-245 in number, and 85-100% of recovered larvae were from their 1iver tissues. 4. Toxocara canis larvae, 45, 31, 42 and 23 in number at 3rd, 14th, 25th and 55th day in one-half of the tissues and organs after infection respectively, were demonstrated from the mice infected with 500 larvae collected from infected chicken liver. Most of the larvae were recovered from the carcass of the mouse. It was approved the larvae isolated from chicken possess infectivity to the mice. 5. Typical eosinophilic granulomatous change was not observed in the liver tissue of the infected chicken at 20th day after infection. As it summarized above, the liver of various domestic animals is the favorite tissue for migration of nematodes larvae. Therefore, the possibility of human visceral larva migrans may be induced due to eating of raw liver of domestic animals.

[Immobilization-Agglomeration Reaction For The Diagnosis Of Trichomonas Vaginalis Infection]

The Trichomonas immobilization-agglomeration reaction was studied using the sera from women with vaginal trichomoniasis and from rabbits inoculated with cultures of T. vaginalis. It was found that the greatest amount of immobilization occurred at 25 to 30 minutes, and that inactivation of the sera did not affect the results. An evidence of antigenic differences between T. vaginalis, T. hominis and C. albicans was confirmed. The immobilization reaction was positive in 80.3% of the 71 T. vaginalis positive cases, but in only 7.7% of the 77 persons of T. vaginalis negative cases. From the above results, it is suggested that immobilization reaction is a useful tool for the diagnosis of T. vaginalis infection.