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.

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.

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) 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.

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 percent) 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.

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: 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). 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. 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. 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. 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.

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.

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 percent 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 percent 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 percent 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. 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 percent out of l,048 inhabitants, and its rate was higher markedly in male(57.7 percent) than in female (15. 1 percent), and the highest rate among the group of 31-40 years old. Eating rate of raw liver of fowl was 5.9 percent, pig 5.3 percent, and dog 2.5 percent. Larva recovery rate from beef liver was 11.8 percent out of 195 samples and 72.0 percent of total detected 1arvae were identified as Toxocara(=Neoascaris) vitulorum. From pig liver, larvae of nematoda were found in 6.4 percent out of 109 samples but no larva was detected from 120 fowl livers. 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 percent of recovered larvae were from their 1iver tissues. 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. 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.

Parasitological Studies on Liver Abscess in Cheju Island

Clinico-pathological, parasitological, microbiological and immunologic studies were made in 238 of islanders Cheju, a highly endemic area of amebiasis in Korea. Ameba immobilization test was carried out using immunized rabbit sera and several strains of Entamoeba histolytica. The results are summarized and concluded as follows; I. The number of males with liver abscess was markedly greater than female, and the largest group distribution was observed in ages 30-39. In liver abscess patients, an increase of white blood cells(W.B.C.) and a decrease of red blood cells'R.B.C.) was noted. The differential count of W.B.C. showed an increase of segmented neutrophils and a decrease of 1ymphocytes. The level of total cholesterol and its ester was also diminished, and urobilinogen in patients, urine was increased in comparison with healthy controls. 2. The detection rate for microorganisms in aspirated liver abscess was 42.1%. Most of the recovered microorganisms were Gram negative enteric bacilli; Escherichia coli, Alkaligenes fecalis, Aerobacder aerogenes and paracolon group. Fungi and cocci were not observed. 3. In the immobilization test using immune rabbit sera immunized with a 48-hour-culture of E. histolytica, the highest immobilization reaction occurred 45-105 minutes after the beginning of the test and remobilization of the parasite took place gradually. Immobilization of ameba continued for more hours and at higher rate in the inactivated rabbit sera group, and the differences among ameba strains were not remarkable. In human amebiasis sera, the highest peak of immobilization reaction occurred at 45-90 minutes after testing with the parasite. 4. Positive rates for the immobilization test according to clinical feature were 83.3-100% in liver abscess cases, 83.3-90.7% in hepatomegaly cases, 45.4% in asymptomatic cyst-passers and 31.5% in healthy controls. 5. For 56 patients who showed a high rate of immobilization, the follow-up positive rate after treatment for amebiasis was markedly reduced in 2-3 months.