• Parasites, Hosts and Diseases
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• v.22 no.2
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• pp.171-180
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• 1984

In order to provide some clues for differential diagnosis of trematode infections in fecal examination, the comparative morphology of eggs of 5 kinds of heterophyid flukes (Metagonimus yokogawai, Heterophyes heterophyes nocens, Heterophyopsis continua, Stellantchasmus falcatus and Pygidiopsis summa) and Clonorchis sinensis was studied. The eggs were obtained from distal portion of uteri of worms which were recovered from men after treatment. The characteristic shape and appearance of each kind of eggs were observed in detail under light microscope, and their length and width measured and compared one another. The results are as follows: 1. Eggs of C. sinensis are elongated ovoidal in shape with attenuated anterior end, 25.3~33. 2 (28. 3 in average) ${\mu}m$ long and 14.2~17.4(5.9) ${\mu}m$ wide with length/width ratio of 1.60~2.00 (1.78). They differ from all heterophyid eggs in that they have prominent wrinkling (muskmelon pattern) at their shell surface. 2. P. summa eggs are ovoid to pyriform in shape and characterized by the smallest size of all kinds examined, 19.8~22.9 (21.6) ${\mu}m$ long and 11.1~13.4 (12.1) ${\mu}m$ wide and the ratio 1.63~1.99 (1.78). 3. Eggs of S. falcatus are elongated ovoidal and most slender form, 25.3~29.2 (27.2) ${\mu}m$ long and 11.1~13.4 (12.5) ${\mu}m$ wide with the ratio of 2.00~2.57 (2.17). 4. Eggs of M. yokogawai are ellipsoid to elliptical in shape with round both ends, 26.9~31.6 (28.5) ${\mu}m$ long and 14.2~18.2 (16.8)${\mu}m$ wide with the ratio of 1.48~2.11 (1.70). 5. H. continua eggs are oval in shape, sometimes similar to M. yokogawai or H. h. nocen$ eggs, however, the relative breadth is broadest among all kinds, with maximum width at posterior half portion. They are 23.7~27.7 (25.0) ${\mu}m$ long, 15.8~18.9 (16.4) ${\mu}m$ wide with the ratio of 1. 33~1.75 (1.53). 6. Eggs of H. h. nocens are ellipsoid to ovoid in shape but sometimes more slender than M. yokogawai and have slightly pointed both ends. They are 23.7~29.2 (25.7) p.m long, 14.2~15.8 (15.4) ${\mu}m$ wide, and the ratio 1.50~2.06 (1.67). From the results, it is concluded that eggs, of 5 kinds of heterophyids and C. sinensis can be morphologically differentiated one another, however, careful observation and measurement on sufficient number of eggs are needed.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.115-120
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• 1986

This study was carried out to reveal the effect of temperature, salinity and aeration on maturation and hatching of Fibricola seoulensis eggs. The eggs were incubated and were observed daily for the appearance of eyespots and hatching. The results were summarized as follows. 1. From $4{\sim}5$ days after incubation in distilled water at $28^{\circ}C$ or at $11{\sim}26^{\circ}C$, the eyespots began to appear and the rates of eggs with eyespots were over 90% in $28^{\circ}C$ on the 7th or 8th day. However, eyespots did not appear in $5{\sim}15^{\circ}C$ or $4^{\circ}C$ by the 18th day. 2. The mature eggs began to hatch at the 8th day, and hatching rate 2 weeks after incubation was over 90% at $28^{\circ}C$, but it was below 5% at $11{\sim}26^{\circ}C$, and 0% at $5{\sim}15^{\circ}C$ and at $4^{\circ}C$. 3. Aeration did not influence the appearance of eyes pots nor hatching. 4. In salines under 0.6%, the rates of eyespots appearance were over 90% on the 7th day. The rate was 55.0% in 0.9% at 20 days, and 0% in 1.2%. 5. The hatching rates in salines below 0.3% concentration were over 90% by 14 days of incubation. However, the rate decreased to 44% in 0.6% saline and to 0% over 0.9% salinity. 6. The eggs incubated in the dark hatched in 12.5% on the 10th day, but hatching rate of mature eggs increased to 85.7% within 2 hours after exposure to light. Above results demonstrated that the best temperature for maturation and hatching of F. seoulensis eggs was $28^{\circ}C$, and the miracidia began to hatch at $8{\sim}9$ days after incubation. In the field, hatching and invasion into snails of the miracidia may occur from May to September in Korea. In salines under 0.3% concentration maturation and hatching were not influenced, but as salinity increased hatching was inhibited more than maturation was.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.121-136
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• 1986

Recently there have been some reports on human infections of Echinostoma hortense in Korea. It was found that a few species of freshwater fishes were playing the role of the second intermediate host of E. hortense. However, molluscan intermediate host has not been identified yet in Korea. The present study aimed to establish the life cycle of E. hortense in laboratory. Experimental studies such as egg production from the rat, development of the eggs in vitro, exposure of miracidia to freshwater snails, shedding pattern of cercariae from infected snails, morphology of cercariae, cercarial infection to the second intermediate host and infection of metacercariae to the definitive hosts were done. In addition, epidemiological surveys on the infection status in inhabitants and house rats, and on the natural infection of larval echinostomes in the snails and fishes were carried out along the South Hangang-river. The results obtained were as follows: 1. The eggs deposited from adults in physiological saline were cultivated at room temperature($20{\sim}24^{\circ}C$). The miracidia were firstly observed on 8 days after cultivation, and 85.5% of the eggs contained the mature miracidia on 11 days after cultivation. More than 90% formed the miracidia when cultivated at temperature $22{\sim}27^{\circ}C$. Hatching of the miracidia began on 12 days after cultivation and continued for a week. The size of the miracidia was $103.0{{\times}}51.4{\mu}m$ in average. The motility of the miracidia were active up to 8 hours after shedding, but they were all dead within 10 hours after shedding. 2. A freshwater snail, Radix auricularia coreana was cultivated in aquaria. A hatched $F_1$ snails from the egg masses were exposed to 20 miracidia respectively. Escape of cercariae started on 15 days after infecton. Radix auricularia coreana was experimentally identified as the first intermediate host of E. hortense in Korea. 3. Cercarial shedding started on $15{\sim}20$ days after infection by snail, continued for about 10 days (8.8 days in average). Infected snails were dead within 32 days after the miracidial infection. About 1,335 cercariae($328{\sim}1,994$) per snail were shed in its life, and 119 cercariae in average per snail per day were shed. The cercariae were motile for more than 24 hours, and then squirming at the bottom until death. The body and tail sizes of cercariae were $356{\times}186{\mu}m$ and $510{\times}68{\mu}m$ in average, respectively. The rediae parasitized in the snail hosts were found mainly around the pericardial regions, and their size was $1,575{\times}258{\mu}m$ in average. The numbers of developing cercariae in a mature redia were 14 in average ($7{\sim}20$ in range). The numbers of rediae in a snail were 102 in average on 15 days after miracidial infection and 221 in average on 28 days. 4. Three uninfected Misgurnus anguillicaudatus, less than 6.5cm long were used in for the cercarial infection. They were all exposed with 755 cercariae, and examined at 5-day intervals starting from 10 days after infection. All the fishes were infected with metacercariae of E. hortense and a total of 275 was found infected(36.4%). The metacercariae were fed to rats and the adult worms were obtained on 15 days after infection. 5. The infected rats began to deposit the eggs on 11 days after infection. The number of eggs deposited per day per worm (EPD/worm) was $400{\sim}500$ on 3 weeks after infection and was increased to $1,000{\sim}1,500$ on 4 to 17 weeks, then decreased to 800 on 21 weeks after infection. 6. A total of 745 stool specimens collected from 576 male and 169 female residents of 8 different villages along South Hangang basin was examined. Out of 745 specimens, the eggs of Echinostoma sp. were found in 2 cases (0.3%). Of 34 house rats one showed egg-positive (2.9%). 7. Total 971 Radix auricularia coreana collected from 7 sampling stations were examined for shedding of cercariae. Three snails (0.3%) shed the cercariae of E. hortense. A total of 119 out of 542 freshwater fishes (22.0%) had the metacercariae of E. hortense. The fishes parasitized with the metacercariae were 4 out of 14 examined species. The infection rates of 4 species were 34.1% (106 out of 311) in Misgurnus anguillicaudatus, 30.4% (7 out of 23) in Misgurnus mizolepis, 4.3% (2 out of 46) in Moroco oxycephalus and 22.2% (4 out of 18) in Odontobutis obscura interrupta. In summarizing the above results, the first intermediate host of E. hortense was found as Radix auricularia coreana in Korea. Also, it took about 46 days for the shortest completion of a life cycle of E. hortense in summer; that is, 10 days for miracidial development in eggs, 15 days for cercarial development in the snail, about 10 days for metacercarial development in the second intermediate hosts, and 11 days for the maturation as the adults in the definitive hosts. The natural infection rates of E. hortense in the intermediate hosts were relatively high but those in the definitive hosts were low in the middle areas of South Hangang basin.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.137-144
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• 1986

The author has studied the $\beta$-glucuronidase activity in several tissues such as liver, stomach and small intestine of the male and female rabbits infected with different doses of metacercariae of Clonorchis sinensis. The metacercariae of Clonorchis sinensis were isolated from Pseudorasbora parva caught in Kim Hae by digestion technic. The experimental animals were sacrificed in the period of 1, 7, 14, 21, 28 and 35th days following the infection. The results obtained were summarized as follows. 1. In the groups infected with 100 metacercariae, $\beta$-glucuronidase activity was slightly increased during the entire periods than control rabbits. It was the highest in the first day with 1.535 and $1.421m{\mu}/g$, 14th days with 2.521 and $2.200m{\mu}/g$, and then lowered by the time, gradually. 2. In the groups infected with 500 metacercariae, $\beta$-glucuronidase activity was highly increased on the first day with 1.535 and $1.856m{\mu}/g$ than that 100 metacercariae groups according to each organs. It was the highest on the 7th day and 14th day. 3. In the groups infected with 1,000 metacercariae, $\beta$-glucuronidase activity was remarkably increased in the first and 14th days according to each organs, and then lowered gradually day by day. 4. $\beta$-glucuronidase activity of all organs was more increased than that of normal organs and the highest activity in the liver with $2.521m{\mu}/g$, intestine(1.612) and stomach (1.581) respectively. 5. $\beta$-glucuronidase activity of rabbits was higher in the female than in the male. On the basis of these results, it was suggested that $\beta$-glucuronidase activity was affected by the duration of infection and by the number of Clonorchis sinensis, according to the organs and sex of the rabbits.

• Parasites, Hosts and Diseases
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• v.29 no.2
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• pp.113-120
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• 1991

When the component proteins in crude saline extract of 13-week old adult Paragonimus westermani were observed by non-denaturing discontinuous-polyacrylamide gel electrophoresis (Disc-PAGE), 8 distinct bands were clearly recognized. Molecular weight (MW) of each band protein, numbered in sequence from cathodal side which appeared in 10% separating gel, was measured first by Ferguson plot utilizing different gel concentrations from 10% to 4.5%. MW of band 1 Protein (known as egg Protein) was 440 kDa. And MW of other band Proteins were: 386 kDa in band 2, 17.4 kDa in band 3, 17kDa in band 4, 14.3 kDa in band 5, 46 kDa in band 6, 38 kDa in band 7 and 23 kDa in band 8. When the proteins in the crude extract were separated into fractions by molecular sieve chromatography through 1.6 (Φ)×70cm sired Sephacryl 5-300 Superane column and revisualized by Disc-PAGE in 8% gel, the sequence of fluted proteins was band 1, band 2, band 6, band 7 and bands 3,4,5 and 8. This elusion profile confirmed MW of each band protein in the crude extract as measured by Ferguson plot.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.145-158
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• 1986

This study was undertaken to purify cystic fluid (CF) antigen of Taenia solium metacestodes by affinity chromatogaphy using specific monoclonal antibody(McAb) and to characterize the antigenicity of the purified antigen. The hybridoma cell lines, prepared by fusion between mouse plasmacytoma and spleen cells from BALB/c mice immunized with CF, secreted antibodies reacting to various helminthic antigens. Majority of cell lines reacted to CF only but some also reacted to parenchymal antigen of T. solium metacestodes, adult T. saginata, sparganum, hydatid cystic fluid, Paragonimus westermani and Clonorchis sinensis, either in combination with CF, other antigens or independently. Cloned cells derived from monoclonal lines also produced antibodies reacting either to CF only or to other helminthes in combination or independently. These results indicated that CF of T. solium metacestodes contained proteins which possessed antigenic determinants not only specific to CF but also cross reactive with the afore-mentioned helminthes. CF of T. solium metacestodes was purified by affinity chromatography using the McAb which reacted to CF and parenchymal antigens. The affinity-purified antigen (A-Ag) and unbound pool CF (U-Ag) were separated. A-Ag showed 2 protein bands by disc-PAGE whereas CF exhibited 6 bands and U-Ag consisted of all bands CF had. The diagnostic significance of A-Ag was evaluated by ELISA in human neurocysticercosis and other helminthic and neurologic diseases. By A-Ag, the levels of the specific IgG antibody, as shown by absorbance in sera and CSF, were lower than those of CF and U-Ag. Accordingly, the sensitivity was about 70% of CF and U-Ag. However, the nonspecific positive reactions to CF and U-Ag, observed in sparganosis, T. saginata infection and paragonimiasis did not occur when A-Ag was used. These results indicated that the affinity-purified A-Ag had the higher specificity but the lower sensitivity as a diagnostic antigen in cysticercosis, probably because it only detected a single or limited numbers of monospecific antibodies among the diverse polyclonal antibodies produced in the patients with neurocysticercosis.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.159-170
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• 1986

A total of 69 patients of confirmed neurocysticercosis was followed serologically by ELISA up to 22 months after praziquantel treatment. The intervals and numbers of follow-up were variable by patient. Serially collected samples of serum and CSF were examined simultaneously for their specific IgG antibody levels by ELISA, using cystic fluid, saline extracts of bladder wall and scolex as antigen. Within 4 months after praziquantel treatment, the antibody levels were elevated temporarily in both serum and CSF in most patients. In some cases antibody levels exhibited steady declining tendency after the treatment. Concomitant administration of dexamethasone appeared to suppress the elevation of antibody levels. The rate of mean absorbance of antibody changed more in serum than in CSF. The rate of elevation was greater in antibodies to parenchymal antigens than that to cystic fluid, but absolute difference of antibody levels was greater in antibody to cystic fluid. Previously negative samples for IgG antibody may become positive after the praziquantel treatment, which could be used as a complementary tool (provocation test) in serodiagnosis. One month was considered to be sufficient interval for the follow-up test for that purpose. In the follow-up of up to 22 months, only few cases of chronic neurocysticercosis showed declining tendency of IgG antibody levels below negative range. During acute encephalitic attacks in chronic patients, IgG antibody to parenchymal antigen were elevated in CSF temporarily. These results indicated that serologic follow-up of every year was recommendable to differentiate the cured patients from chronic patients with slowly calcifying lesions.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.187-193
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• 1986

Enzyme-linked immunosorbent assay(ELISA) using crude and affinity-purified antigens of adult worms of Paragonimus westermani was performed for infected cat sera with different worm burden, from preinfection to 18th week after infection. Crude antigen was used with supernatant of homogenated worms by freezing-thawing method, and the supernate was centrifuged for 1 hour at 10,000 rpm at $4^{\circ}C$. Affinity-purified antigen(antibody-bound antigen) was prepared from fractions(bound and unbound) of crude antigen by affinity chromatography on CNBr-activated sepharose 4B, and IgG as a ligand was prepared from paragonimiasis cat serum(6 months infected) obtained by ammonium sulfate ($40%{\sim}45%$ saturated) precipitation method. By SDS-PAGE, crude antigen showed 22 polypeptide fractions while purified antigen showed 4 fractions: 36, 400, 34, 700, 27, 600 and 11, 500 in molecular weights. All cats were divided into five groups($G_1-G_5$) by different worm burdens. The mean of recovered worms(${\pm}SD$) and the number of cats in each group are as follows: $G_1$, 2 worms(0) and 4 cats; $G_2$, 4.75 (${\pm}0.66$) and eight; $G_3$, 10.75(${\pm}1.92$) and four; $G_4$, 23.20(${\pm}3.43$) and five; $G_5$, 48(${\pm}12.63$) and five cats. The results were summarized as follows: 1. The antibody levels(OD value) increased by worm burden in $G_1$ to $G_4$ generally. However, individual antibody levels were not exactly related with worm burden in all groups, especially there was a wide difference in $G_4$ and $G_5$. These results suggested that the worm burden in $G_4$ (about $20{\sim}30$ worms) is enough to produce antibody maximum in cats of $2{\sim}3kg$ weight. 2. The antibody levels increased significantly(p<0.05) compared to control sera at the 3rd week in $G_1$ and $G_2$, at the 2nd week in $G_3$, and at the 1st week in $G_4$ and $G_5$. Especially in the 4th week, OD value increased more in $G_1$(p<0.01) and in $G_2$ to $G_5$(p<0.001). In the pattern of antibody levels by ELISA in each group, OD in $G_1$ increased to the 18th week continuously, in $G_2$ OD was maintained same after the 16th week, but in $G_3$ it decreased after the 16th week, and it was maintained same in $G_4$ and $G_5$ after the 14th week. 3. The antibody levels by ELISA with the affinity-purified antigen were higher than those with crude antigen in all groups generally. Especially, the difference of OD values between two antigens was larger from the 4th to the 10th week. In $G_1$ and $G_2$ OD with purified antigen was higher than that with crude one to the 18th week. It was also higher in $G_3$ than that with crude antigen to the 16th week and OD of $G_4$ and $G_5$ were higher before the 14th week than that with crude antigen, however became lower at the 16th week. Consequently, the antibody level in ELISA with affinity-purified antigen was more sensitive at the early weeks after infection and in light infection groups than that with crude antigen.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.109-114
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• 1986

In order to observe the growth and development of Fibricola seoulensis metacercariae, the tadpoles of Rana nigromaculata were experimentally infected with the cercariae. The meta cercariae of various developmental stages were recovered from the tadpoles after 2 to 65 days of infection. They were prepared for morphological observation, and were given orally to mice to observe their infectivity. The following results were obtained. 1. All of the tadpoles exposed to the cercariae were observed to harbour the larvae in their abdominal cavity. 2. The young metacercariae of 2 days after infection were $121.1{\mu}m$ long and $63.3{\mu}m$ wide. They grew linearly for the first 14 days to be $262.0{\mu}m$ long and $166.4{\mu}m$ wide. Thereafter, no more growth recognized until 65 days. 3. The larvae of 2 days old were similar with cercarial body and had 2 suckers, a pharynx, 2 ceca and a primordium of germ cells but no tribocytic organ. On the 8th day, they had tribocytic organ, and their morphology resembled that of mature metacercariae. 4. The metacercariae younger than 10 days could not infect the mice. Only the metacercariae older than 14 days had infectivity. The recovery rates increased by the age of metacercariae from 19.0% in 14 days old to 70.0% in 40 days old. Above findings indicate that the tadpole is indispensable for metacercarial development and it needs at least 2 weeks for maturation. The tadpole is a pivotal host in the life cycle of F. seoulensis for connection between the snail and the frog.

• Parasites, Hosts and Diseases
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• v.36 no.4
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• pp.255-260
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• 1998

To evaluate the biological and biochemical characteristics of Trichomonas vaginalis KT9 isolate, the growth and size of trichomonads, pathogenicity in mouse, protein profiles and proteinase activity were examined after shifting the medium from TPS-1 into TYM. Generation time of trichomonads in TYM medium was 4.5 hr in comparison to TPS-1 with 7.1 hr. Size of trichomonads cultured in TPS-1 medium ($8.5{\;}{\pm}{\;}0.9{\;}{\times}{\;}6.0{\;}{\pm}{\;}0.9{\;}{\mu\textrm{m}}$) was significantly smaller than those in TYM medium ($10.9{\;}{\pm}{\;}1.4{\;}{\times}{\;}8.2{\;}{\pm}{\;}0.9{\;}{\mu\textrm{m}}$). Trichomonads cultured in TYM medium produced subcutaneous abscess in 9 out of 10 mice, whereas those in TPS-1 medium produced abscesses in 2 out of 10 mice. In SDS-PAGE, trichomonad Iysates from both media showed ten common bands. However, trichomonads in TYM medium showed additional bands of 136 kDa, 116 kDa and 40 kDa in comparison to those in TPS-1 with 100 kDa. By immunoblot with T. vaginalis-immunized rabbit sera, T. vaginalis cultivated in both TYM and TPS-1 media showed 5 common bands. and unique bands of 116 kDa. 105 kDa. and 86 kDa were observed in trichomonads in TYM while a 140 kDa band in those in TPS-1. In gelatin SDS-PAGE, trichomonads in TYM degraded gelatin stronger than those in TPS-1. Also protease activity of trichomonads in TYM was significantly higher than that of trichomonads in TPS-1 using Bz-Pro-Phe-Arg-Nan as a substrate. According to the results, it is assumed that the shift from TPS-1 into TYM medium for cultivation of T. vaginalis might modulate the biological and biochemical properties of T vaginalis in vitro.