Molecular and electrophysiological characterization of nucleotide-sensitive chloride current-inducing protein of Fasciola hepatica.

Nucleotide-sensitive chloride current regulating proteins (ICln's) of the chloride channels have been characterized from man and animals. An ICln of Fasciola hepatica (ICln-Fh) consisting of 231 amino acids revealed high similarities to both consensus domain of ICln's and two acidic residue-abundant patches in its C-terminus. Native ICln-Fh protein was confirmed present in F. hepatica soluble extract by immunoblotting. The recombinant ICln-Fh protein expressed in collagenase-defolliculated Xenopus oocytes induced fast rising and outward rectifying Cl- currents (I(Cln-Fh)). The recombinant ICln-Fh protein, however, did not trigger cell swelling-induced Cl- currents (I(Cl-swell)). The I(Cln-Fh) currents were significantly reduced by substituting external Cl- with gluconic acid and by externally adding cAMP. Collectively, these results suggest that ICln-Fh protein is an inducer of Cl- currents in F. hepatica.

Use of a recombinant Clonorchis sinensis pore-forming peptide, clonorin, for serological diagnosis of clonorchiasis.

A recombinant pore-forming peptide of Clonorchis sinensis, clonorin, was evaluated for serodiagnostic reagent of clonorchiasis by enzyme-linked immunosorbent assay detecting IgG antibody. Recombinant clonorin showed 100% specificity and low sensitivity for sera of human clonorchiasis. In contrast, C. sinensis crude antigen revealed lower specificity and higher sensitivity than recombinant clonorin did. In sera of experimental rabbits, clonorin-specific IgG antibody was increased remarkably 8 weeks after the infection and retained around level of OD(490)=0.2 for 1 year. With excellent antigenic specificity, it is suggested that the recombinant clonorin can be used as an ingredient of the cocktail antigen for serodiagnosis of clonorchiasis from early stages of the infection.

Molecular cloning and enzymatic characterization of a class mu glutathione S-transferase of Paragonimus westermani.

Glutathione S-transferase (GST) is a component of a second line of defense against bioreactive radicals derived from host immune attack. Paragonimus westermani causes acute or chronic lung diseases in mammals. A cDNA clone, PwGST#11, of adult P. westermani produced in the present study was 748 bp long and encoded an open reading frame of 217 amino acids with a starting methionine. The molecular mass of this putative polypeptide, Pw26GST, was estimated to be 25.1 kDa with an isoelectric point of 5.7. Pw26GST was homologous with the 26-kDa GSTs of trematodes and vertebrates. Nine of the ten amino acid residues lining the glutathione-binding pocket were conserved. Putative Pw26GST polypeptide was clustered with 26-kDa GSTs of trematodes belonging to the class mu. Recombinant Pw26GST protein generated bacterially, revealed GST enzyme activity toward an universal and class mu-specific substrates. Mouse antisera to recombinant Pw26GST protein recognized native 26-kDa GST of P. westermani but not the GSTs of any other trematodes. Collectively, Pw26GST was found to be a member of class mu GSTs of P. westermani.

Expressed sequence tag analysis of adult Clonorchis sinensis, the Chinese liver fluke.

Expressed sequence tag (EST) pools represent partial profiles of the gene expressions of organisms. In an effort to construct a Clonorchis sinensis EST pool, 2,387 ESTs were collected from an adult C. sinensis cDNA library and assembled into 1,573 clusters. Of these clusters, 1,225 ESTs (51%) were singletons and 348 clusters consisted of more than two ESTs. There were 848 clusters (54%) that shared significant identity with previously reported proteins, and of these, 401 clusters were categorized into 11 major functional protein classes. Three cDNA clones of fructose-1,6-bisphosphate (FBP) aldolase were selected from the C. sinensis EST pool and analyzed for phylogenic clustering. FBP clones encoded a complete polypeptide, which shared significant identity to those of vertebrate and invertebrate animals and clustered with those of trematodes. We believe that the EST pool described can be confidently used as a platform in multigene researches on C. sinensis gene expression.

Identification of immunodominant excretory-secretory cysteine proteases of adult Paragonimus westermani by proteome analysis.

Paragonimus westermani causes inflammatory lung disease in humans. The parasite excretes a host of biologically active molecules, which are thought to be involved in pathophysiological and immunological events during infection. Analyses of the 2-DE protein profiles of the excretory-secretory products (ESP) of adult P. westermani revealed approximately 147 protein spots, at least 15 of which were identified as cysteine proteases (CPs), at pHs between 4.5 and 8.5, and molecular weights (MWs) between 27 and 35 kDa. An additional three CPs (designated as PwCP-3, -8 and -11) were newly recognized by TOF/TOF MS. Their molecular biological information, which shared a high level sequence homology, was elucidated. The majority of the CPs reacted strongly with sera from paragonimiasis patients. When we observed the chronological changes in the antibody responses of the respective CPs against canine sera collected serially at 1, 3, 5, 7, 11 and 14 wk after experimental infection, these molecules exhibited a multiplicity of distinct immune recognition patterns. Our results clearly showed that P. westermani adult ESP were principally composed of excretory-secretory CPs, and that these CPs may exert effects not only on host tissue degradation and nutrient uptake, but also on the immune-regulating cells via synergistic and independent interactions.

A human infection of Echinostoma hortense in duodenal bulb diagnosed by endoscopy.

As gastroduodenoscopy performed more frequently, case reports of human echinostomiasis are increasing in Korea. A Korean woman presented at a local clinic with complaints of abdominal pain and discomfort that had persisted for 2 weeks. Under gastroduodenoscopy, two motile flukes were found attached on the duodenal bulb, and retrieved with endoscopic forceps. She had history of eating raw frog meat. The two flukes were identified as Echinostoma hortense by egg morphology, 27 collar spines with 4 end-group spines, and surface ultrastructural characters. This report may prove frogs to be a source of human echinostome infections.

Significance Of Scotch-tape Anal Swab Technique In Diagnosis Of Enterobius Vermicularis Infection.

The significance of Scotch-tape anal swab technique was evaluated in three communities of Korea, one in orphanage institute and two in rural populations, from November to December, 1975. Based on the epidemiological concept that the prevalence rate of Enterobius vermicularis infection in a community as "the proportion in the population who harboured E. vermicularis at certain point of time", the present authors treated the whole surveyed population with pyrantel pamoate disregard to the results of Scotch-tape anal swab and collected pinworms expelled in stool specimens during 2 consecutive days after the chemotherapy. Although the present authors could not collect the younger adult worms less than 3.54 mm in length after chemotherapy, the positive rates of pinworm collection in three surveyed communities were 80.6%, 92.5% and 91.4% respectively whereas the positive rates of single Scotch-tape anal swab were 52.4%, 53.6% and 57.1% respectively. These results denote that results of single anal swab do not represent the prevalence rate of Enterobius infection in a community. The results of successive two anal swabs and estimation of positivity in a population using Neyman's "Best asymptotically normal estimate" revealed 62.9% in the third trial group of this study and probability of finding eggs in single slide was 0.869. Comparing with the pinworm collection rate after the chemotherapy in this group the estimated positive rate was by far lower than that of pinworm collection(89.3%). The positive results of single anal swab did not correspond to the pinworm collection in average 9.1% of anal swab positive cases and the negative results did not correspond to pinworm collection in 81.3% of anal swab negative cases, when the data from three surveyed communities were amalgamated. These results must come from the principle of anal swab that detect the terminated parasitism. With rare exceptions, the anal swab negative cases harbour relatively fewer number of Enterobius than those of positive cases. And the mean number of E. vermicularis collected from anal swab negative cases was 9.1 whereas the number in anal swab positive cases was 31.5. By analyzing the data on the relationship between bathing interval and anal swab positive conversion, it was assumed that the positive rate of anal swab in a community represent the rate of appearance of gravid female Enterobius vermicularis through anus during approximately past two days prior to examination.

Specific IgG antibody responses in experimental cat metagonimiasis.

In order to observe the feasibility of serologic diagnosis of metagonimiasis, saline extracts of metacercariae and 4-week old adults were prepared. Sera from 25 experimentally infected cats were collected from 3 days to 12 weeks after infection. Their levels of specific IgG antibody were measured by ELISA together with 3 sera from non-infected cats. Specific IgG antibody levels began to rise in 7 days after infection, reached their peak in 2-4 weeks and made a plateau thereafter. Cats infected with hundreds of adult worms showed minimal rise of the antibody level. Adult antigen was superior to metacercarial antigen in detecting the specific IgG antibody.

Analysis of antigen specificity using monoclonal and polyclonal antibodies to Cysticercus cellulosae by enzyme-linked immunoelectrotransfer blot technique.

To analyse the antigen specificity of patients sera from 24 confirmed neurocysticercosis and a monoclonal antibody, SDS-PAGE using 10-15% linear gradient gel and EITB were done. Cystic fluid, saline extracts of scolex and of whole worm of C. cellulosae, saline extracts of sparganum, hydatid cyst fluid, saline extracts of Fasciola, Clonorchis and Paragonimus were used as antigen. Of protein bands in cystic fluid of C. cellulosae, patient sera reacted frequently to bands of 152, 94, 64, 48, 24, 15, 10 and 7 kDa proteins. To saline extracts of scolex and whole worm of C. cellulosae, patients sera reacted frequently to 94, 64, 52, 39, 34, 15 and 10 kDa bands. Two bands in sparganum extract (130 and 64 kDa) and two bands in hydatid cyst fluid (52 and 27 kDa) were cross-reacting bands with sera from cysticercosis patient. Saline extracts of Fasciola, Clonorchis and Paragonimus did not exhibit cross-reacting bands. Monoclonal antibody to cystic fluid of C. cellulosae was found to react with low molecular weight proteins of 15, 10 and 7 kDa.

[On The Applicability Of Partially Purified Antigenic Preparations Of Paragonimus Westermani]

In order to obtain more specific antigenic preparation for the diagnosis of human paragonimasis, crude saline extract of whole worm (=PwWWE), secretory-excretory components (PwSEC) and secretion-excretion-free somatic extract (PwSM) of 12 week-old Paragonimus westermani were filtrated through Sephadex G-200 gel column. The adult Paragonimus worms were obtained from experimentally infected dogs. A total of 11 antigenic solutions was lyophilized or diluted to adjust protein content of 1 mg/ml. To evaluate the antigenicity of crude antigens and fractions, micro-ELISA was done with the sera from P. westermani infected cases, C. sinensis infected cases and non-infected control cases to detect Paragonimus specific IgG antibody. The results were as follows: When the PwWWE was filtrated through Sephadex G-200 gel, it was separated into three fractions; PwWWE Fr. 1, PwWWE Fr. 2 and PwWWE Fr. 3. The percentage of protein content was 28.0 %, 21.6 % and 50.4 % respectively. The PwSM was also separated into three fractions; PwSM Fr. 1, PwSM Fr. 2, PwSM Fr. 3 and their percentage of protein content was 41.3 %, 38.6 % and 20.1 %. However, the PwSEC showed different fractionation pattern; i.e. fraction 1 (=PwSEC Fr. 1) and 3 (PwSEC Fr. 3) without fraction 2. The percentage of protein content was 14.0 % in PwSEC Fr. 1 and 86.0 % in PwSEC Fr. 3. When the antigenicity of each Paragonimus crude antigen and fractionated antigen was evaluated for specific IgG antibody by micro-ELISA in 10 human paragonimiasis sera, PwSEC Fr. 1 was the most potent antigen showing the mean absorbance 1.98. The PwWWE Fr. 1, PwSEC, PwWWE were next to that; their mean absorbance were 1.72, 1.38 and 0.83, respectively. The antigenicity of fractions 2 and 3 was much weaker in binding specific IgG antibody. When the antigens were reacted in micro-ELISA with 10 human clonorchiasis sera, most antigens showed weak reactivity. Each fraction 1 of crude antigens reacted higher than other fractions or crude antigens; the mean absorbance was 0.17 in fraction 1, but in others the absorbances were about 0.06. With non-infected control sera, the result of micro-ELISA revealed almost same pattern with those of the clonorchiasis sera. From the above results, it became apparent that PwWWE Fr. 1, especially PwSEC Fr. 1 was the most potent antigen reacted with Paragonimus specifc IgG antibody.

Seasonal variations of metacercarial density of Clonorchis sinensis in fish intermediate host, Pseudorasbora parva.

The seasonal variations of the rate and intensity of metacercarial infection of C. sinensis in P. parva were observed. The fish were collected at Sun-Am river which located in Kim-Hae City, Kyong-Sang-Nam Do(=Province), Korea, from March 1983 to February 1984 every month. A total of 788 fish was examined. The number of metacercariae in each fish was individually counted after the individual digestion by artificial gastric juice. The result was as follows: During one year, 513(65.1%) out of 788 fish were infected with metacercariae. In May, June, July and September, the infection rates ranged from 82. 0 % to 98. 6% whereas the rates was relatively low in March, April, November and February raning from 11. 4% to 64.7%. The intensity of infection was similar with those of infection rates. The mean intensity per infected fish was 103.0 and standard deviation was 118.9 throughout one year. The highest mean intenstiy was in June(294. 8) and the lowest in Novebmver(11.1). The observed frequency of fish with certain intensities of metcercariae were fitted to theoretical equations derived from negative binomial distribution in March, April, November and February(p > 0.05). Meanwhile, the equation of lognormal distribution were fitted with the observed frequencies in May, June, July and September(p > 0.05, p > 0.75). The variance/mean ratio varied by month. The value was the highest in July(814.3) and the lowest in November(158.8). Unlike our hypothesis, the metacercarial density of Clonorchis sinensis in its the most favourable fish host, Pseudorasbora parva showed considerable seasonal variations in the hyperendemic area. The possible factors were discussed.

[A Mathematical Approach To The Mode Of Transmission Of Clonorchiasis In The Inhabitants Of Nak-Dong And Han River Basin]

To understand the mode of transmission in clonorchiasis, a survey was made in Kim-hae Goon, South Kyong-sang Do (=Province). The mathematical analysis of the age prevalence was done on the egg positive rates. And another analysis for the comparison was also made to the cited data from two areas, North Kyong-sang Do and Ko-yang Goon, Kyong-gi Do. Some catalytic models of H. Muench (1959) were applied to the observed age prevalence. Because the both parameters, such as force of infection(a) and loss of positivity(b) were considered to be constant for a long period in the surveyed area, the two stage catalytic model by Muench was chosen to the analysis. In the surveyed area, Kim-hae Goon where the egg positive rates were 56.2% and 61.2% (by Kim, 1974), the constant values of 'a' were found to be 0.051 and 0.089 respectively. In other words, the force of infection was 51, 89 per 1,000 susceptibles. The values of 'b' were found to be 0.006 and 0.005. This means that the rates of disappearance from egg positive cases to negative were 6 and 5 per annum per l,000 positive cases in above area. Therefore, the two catalytic curves were expressed by the following equations, y = 1.133 {e(-0.006t) - e(-0.051t)} and y = 1.047 {e(-0.005t) - e(-0.089t)} respectively. In the cases of North Kyong-sang Do and Ko-yang Goon, Kyong-gi Do where the egg positive rates of clonorchis shown as 27.7% and 15.2% by Shin (1964) and Kim (l974), the curves were expressed by y = 1.769 {e(-0.010t) - e(-0.034t)} and y = 2.857 {e(-0.020t) - e(-0.027t)} respectively. From the above mathematical analyses by age prevalence in clonorchiasis, it was considered that the mode of transmission of clonorchiasis in the surveyed area, Kim-hae Goon presented more rapid pattern than those of North Kyong-sang Do and Ko-yang Goon, Kyong-gi Do.

[Study on the frequency distribution of the metacercarial density of Clonorchis sinensis in fish host, Pseudorasbora parva]

Since the pattern of the frequency distribution of a parasite within a host showed an overdispersed pattern, various statistical models such as Poisson, negative binomial and lognormal distributions have been applied on the population dynamics in host-parasite relations. The observed data on the number of metacercariae of Clonorchis sinensis in a suitable intermediate host, Pseudorasbora parva which were collected from an endemic area, Juk-Rim River, Kim-Hae Goon, South Kyong-Sang Do were applied on the statistical models. The results obtained are as follows. 1. By the calculation of the raw data, 258(94.9%) out of 272 fish showed positive to the metacercarial infection of C. sinensis. The mean number of metacercaria in a fish was 335.1. The standard deviation was 250.6 and the mode was observed between 250 and 299. 2. The frequency distribution pattern of metacercariae of C. sinensis within the fish host in surveyed area was fitted to the lognormal distribution (0.05

Ultrastructural localization of 28 kDa glutathione S-transferase in adult Clonorchis sinensis.

Glutathione S-transferase (28GST) with molecular mass of 28 kDa is an antioxidant enzyme abundant in Clonorchis sinensis. In adult C. sinensis, 28GST was localized in tegumental syncytium, cytons, parenchyma, and sperm tails examined by immunoelectron microscopy. C. sinensis 28GST was earlier found to neutralize bioreactive compounds and to be rich in eggs. Accordingly, it is suggested that 28GST plays important roles in phase II defense system and physiological roles in worm fecundity of C. sinensis.

Studies On The Lungfluke, Paragonimus Iloktsuenensis: IV. A Mathematical Analysis On Metacercarial Density Of Paragonimus Iloktsuenensis In Crab Host, Sesarma Dehaani.

Mathematical models such as the negative binomial, Poisson and Polya-Eggenberger distributions were applied to the observed data of the number of metacercariae of Paragonimus iloktsuenensis in crab hosts, Sesarma dehaani which were collected in Hadong, South Kyong-sang Do, Korea. From the above analysis, it was found that the pattern of density of metacercariae of this lungfluke among the crab hosts was well fitted to the negative binomial distribution, rather than to the Poisson or Polya-Eggenberger distribution.

[Helminthes Infections In The Small Intestine Of Stray Dogs In Ejungbu City, Kyunggi Do, Korea]

One hundred and two stray dogs in Ejungbu City of Kyunggi-Do, Korea were examined to reveal out the degree of natural helminthic infection of small intestine. Helminthes were collected at autopsy, by scraping the intestinal contents. The collected worms were classified by their morphological characteristics. Out of 102 examined, 72 dogs were infected with any helminthes. The common helminthes were Dipylidium caninum (47%), Ancylostoma caninum (26%), Toxascaris leonina (16%) and Toxocara canis (13%). Taenia pisiformis (9%), Echinostoma hortense (4%), E. cinetorchis (2%), Spirometra mansoni (2%) were also found. One dog was incidentally found to be infected with Clonorchis sinensis. The prevalence of Toxascaris leonina was relatively high in this study over Toxocara canis, and its significance was discussed. Dogs were firstly described as the reservoir hosts of Echinostoma hortense and E. cinetorchis in Korea.

Serologic follow-up study in neurocysticercosis patients by ELISA after praziquantel treatment.

/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 patients. 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 anitbody to cystic fluid. Previously negative samples for IgG antibody may become positive after 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.

Intracranial synthesis of specific IgG antibody in cerebrospinal fluid of neurocysticercosis patients.

To determine the source of Cysticercus-specific IgG antibody in cerebro-spinal fluid(CSF), paired samples of serum and CSF were collected from confirmed neurocysticercosis, other neurologic diseases and normal control. The antibody levels in serum and CSF were measured by enzyme-linked immunosorbent assay (ELISA). With the measurement of total protein, albumin and IgG concentration in serum and CSF, the contribution of IgG in CSF were calculated in transudation, exudation and intracranial synthesis using the formula of Tourtellotte and Ma (1978). Mean concentrations of total protein, albumin, IgG and proportional IgG levels in CSF by transudation, exudation and intracranial synthesis were elevated in neurocysticercosis. But only the intracranial synthesis of IgG showed a statistically significant correlation with the specific IgG antibody levels in CSF. In CSF from lateral ventricle in the 4th ventricular neurocysticercosis, the protein concentrations were normal and the specific antibody levels were negative. However, in consecutively secured lumbar CSF from the same patients, the former were increased and the latter were positive. These results indicated that, in neurocysticercosis, the specific IgG antibody in CSF was a local product of intracranial synthesis.

Immunoblot observation of antigenic protein fractions in Paragonimus westermani reacting with human patients sera.

In order to observe the antigenic fractions in saline extract of adult Paragonimus westermani, proteins in the crude extract were separated by sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) in reducing conditions. The separated protein fractions were transferred to nitrocellulose paper on which 20 sera from human paragonimiasis were reacted and immunoblotted. Out of 15 stained protein bands in SDS-PAGE, 7 reacted with the sera. Of 14 reacted bands, 30 kilodalton(kDa) band was the most frequently reacted (95%) and was a strong antigen. Protein bands of 23 and 46 kDa were also strong antigens. Bands of over 150 kDa, 120 kDa, 92 kDa, 86 kDa, 74 kDa, 62 kDa, 51 kDa, 32 kDa, 28 kDa, 16.5 kDa and 15.5 kDa were also reactive but their frequencies of the reaction were variable.

Antigenic protein fractions reacting with sera of sparganosis patients.

To observe the antigenic protein fractions in saline extract of Spirometra mansoni plerocercoid (sparganum), the crude extract was separated in reducing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins, transferred by electrophoresis to nitrocellulose paper, were reacted with sera from 15 surgically confirmed sparganosis and 24 cysticercosis patients for immunoblotting. Out of 30 identified protein bands in the extract, bands of 29 and 36 kilodaltons (kDa) were the strongest and the most frequently reacting with specific antibody (IgG) in sparganosis sera. Bands of higher molecular weight also reacted with the sera but their frequency of reactions was lower. Sera of cysticercosis reacted with different protein bands in saline extract of sparganum, but the cross reactions were observed in strong antigenic bands of 29 and 36 kDa.