Enhanced protection against Clonorchis sinensis induced by co-infection with Trichinella spiralis in rats.

Although co-infection with multiple parasites is a frequent occurrence, changes in the humoral immune response against a pre-existing parasite induced as a result of a subsequent parasitic infection remain undetermined. Here, we utilized enzyme-linked immunosorbent assay (ELISA) to investigate antibody responses, cytokine production and enhanced resistance in Clonorchis sinensis-infected rats (Sprague-Dawley) upon Trichinella spiralis infection. Higher levels of C. sinensis-specific IgG and IgA were elicited upon T. spiralis infection, and these levels remained higher than in rats infected with C. sinensis alone. Upon subsequent infection with T. spiralis, IgG antibodies against C. sinensis appeared to be rapidly boosted at day 3, and IgA antibodies were boosted at day 7. Challenge infection of C. sinensis-infected rats with T. spiralis induced substantial mucosal IgG and IgA responses in the liver and intestine and increases in antibody-secreting plasma cells in the spleen and bone marrow. Subsequent infection also appeared to confer effective control of liver C. sinensis loads, resulting in enhanced resistance. Memory B cells generated in response to C. sinensis infection were rapidly amplified into antibody-secreting cells upon T. spiralis infection. These results indicate that enhanced C. sinensis clearance induced by co-infection is associated with systemic and mucosal IgG and IgA responses.

Immunochemical and biological analysis of allergenicity with excretory-secretory products of anisakis simplex third stage larva.

BACKGROUND: Anisakis simplex third stage larvae (L3) are parasites that frequently give rise to allergic responses. The larvae molt into fourth stage larvae (L4), and at each stage they produce L3-excretory-secretory products (L3-ESP) and L4-ESP, respectively, which are different in their main protein constituents. Although the allergenicity of L4-ESP has been investigated by several research groups, research on the allergenicity of L3-ESP has not been carried out by any researcher. In this investigation, the allergenicity and antigenicity of L3-ESP were investigated in comparison with L4-ESP, using rat sera. METHODS: Rat sera were produced by L3 oral infection two times with a 9-week interval. Larvae ESP prepared by culture were concentrated and fractioned using lyophilizer and a centrifugal filter device, respectively. Immunochemical analysis was performed using both indirect ELISA and immunoblot. Biological allergenicity was analyzed by RBL-2H3 exocytosis. RESULTS: With the indirect ELISA, the optical density (OD) value of the nonfractioned (NF)-L3ESP was only one third of that of the NF-L4ESP in both specific IgM and IgG. On measuring specific IgE, the OD of NF-L3ESP was less than one tenth of that of NF-L4ESP. In addition, neither antigen nor allergen was shown in NF-L3ESP, but it was shown in NF-L4ESP with immunoblot. However, the biological allergenicity of NF-L3ESP was comparable to that of NF-L4ESP. To demonstrate the presence of any allergen, L3-ESP was fractioned and found to carry twelve visualized allergen bands from 10 to 186 kDa by immunoblot. CONCLUSIONS: These results indicate that L3-ESP may include the important allergens necessary to induce the allergy by L3 oral infection, as compared to L4-ESP.

Efficacy of flubendazole and albendazole against Trichinella spiralis in mice.

Efficacy of flubendazole and albendazole against Trichinella spiralis in mice were studied. ICR mice were experimentally infected with Trichinella spiralis and treated with either flubendazole (FBZ) or albendazole (ABZ) at four different stages of the parasite life-cycle. Oral administration of either FBZ or ABZ at 20 mg/kg and 50 mg/kg on 2 h, 8 h and 24 h (pre-adult stage) after infection eliminated 94.7-100% of adults as determined at necropsy on day 7 post infection (p.i.) and 96.9-100% of larvae on day 45 p.i. FBZ was more effective than ABZ against adult T. spiralis (at 2 to 6 days p.i.), when treated with a dosage of 20 mg/kg for 5 consecutive days (99.4% and 46.0% reduction with respect to the control group). Against migrating larval T. spiralis, FBZ was more effective than ABZ at 20 mg/kg for five consecutive days (on days 11-15 p.i.), and the reduction rate of recovered larvae were 99.6% (FBZ) and 80.8% (ABZ) respectively. FBZ was more effective against early encapsulated larval T. spiralis (at 21 to 25 days p.i.), than ABZ when both were given at 20 mg/kg for five consecutive days (99.8% and 45.4% reduction, respectively). In conclusion, flubendazole was more effective than albendazole against adult and parenteral stages of Trichinella spiralis in mice.

Infectivity and pathological changes in murine clonorchiasis: comparison in immunocompetent and immunodeficient mice.

The main complications of clonorchiasis are periportal inflammation, biliary hyperplasia, periductal fibrosis, and subsequently the development of biliary tumors in the liver. This study was undertaken to compare the infectivity and histopathologic changes between in immunocompetent FVB/NJ and BALB/cA strains, and immunodeficient severe combined immunodeficient (SCID) and athymic nude mice after the metacercariae of Clonorchis (C.) sinensis were infected. The experiment showed that C. sinensis was very infective in all strains studies, but the status of worm development, infectivity, recovery rate, and morphological changes of livers were very different in each strain. FVB/NJ mice showed more worm recovery than any other strain. Histopathologically the liver of FVB/NJ mice at 4 weeks postinfection showed marked cystic and fibrotic changes, in which C. sinensis was fully developed with ovum production, severe infiltration of inflammatory cells, mostly eosinophils, and high degrees of biliary hyperplasia. In SCID and nude mice, there were few foci of inflammatory cells even at 8 weeks postinfection in periportal areas of the liver, associated with no development into adult worm with ovum production. Fibrosis occurring at 4 weeks postinfection was highly correlated with inflammatory infiltration when each strain was compared. We suggest that massive infiltration of eosinophil and plasma cells caused by the infection might initiate cystic formation and fibrosis. These data demonstrate that the infection of C. sinensis might be related to pathologic consequences of inflammatory cell infiltration, cystic formation and fibrosis which might play a role in the defense mechanism against the parasitism in the liver of each strain. The FVB/NJ mouse model might be very helpful in elucidating the mechanism for human clonorchiasis.

PCR-RFLP patterns of four isolates of Trichinella for rDNA ITS1 region.

We have studied the genetic differences among four isolates of Trichinella including a new strain of Trichinella spiralis (ISS 623) recently found from a human case who took a badger in Korea. Because they have a different host origin and came from geographically separated regions, we supposed the genetic pattern of the isolates might be different as had been previously reported. It was analysed by PCR-RFLP analysis of the rDNA repeat that can readily distinguish a species or strain from others. Isolated genomic DNA of each isolate of Trichinella larvae was amplified with ITS1 specific primers and digested with restriction endonucleases. The PCR product of ITS1 was confirmed using Southern blot analysis to be a 910 bp fragment. The restriction fragments of each isolate had variable patterns when it was digested with Rsa 1 only. According to the RFLP patterns, the estimated genetic divergence between each isolate was different. In conclusion, four isolates of Trichinella including a new strain of T. spiralis obtained from a Korean patient may have genetic differences in the ITS1 region and the Shanghai isolate was genetically more similar to the Japanese unknown isolate than others in the ITS1 region.

Differentiation of hamster liver oval cell following Clonorchis sinensis infection.

Oval cells which appear in the liver after hepatic injuries are suspected to be progenitor cells for both hepatocytes and bile duct cells. Oval cell isolated from the livers of the hamsters treated with diethylnitrosamine and 2-acetylaminofluorene and infected with Clonorchis sinensis (CS). cultured for 2 weeks and evaluated for differentiation and plasticity by electron microscopy and immunohistochemistry. In the CS-uninfected group, glycogen granules and peroxisomes were noted in the cells that were cultured for 2 weeks. Starting at 1 week postculture, immunoreactivity of the cells to cytokeratin 19 markedly decreased but that to albumin and alpha-fetoprotein gradually increased. This means that oval cells isolated from hamsters that were not infected with CS differentiated toward hepatocyte lineage. However, in the CS-infected group, cultured cells contained numerous rough endoplasmic reticulum and showed immunoreactivity that was generally in reverse to that of CS-uninfected group, meaning that cells isolated following CS infection were primed by CS and differentiated toward bile duct cell lineage. The results of this study suggested that oval cells are indeed bipolar progenitor cells for hepatocytes and bile duct cells and can differentiate toward either lineage depending upon the priming factor.

Chemotherapeutic efficacy of praziquantel in rats with protective immunity to Clonorchis sinensis infection.

OBJECTIVE: To study the synergic effect of praziquantel (PZQ) and host acquired immunity on Clonorchis sinensis. METHODS: Acquired immunity to C. sinensis was induced by immunization with crude adult worm antigen (AW Ag) and excretory-secretory antigen (ES Ag) or infection with C. sinensis metacercariae. The effect was assessed by the worm reduction rate compared with the control groups after challenge infection with 50 metacercariae and treated orally with a subcurative dose of praziquantel (50 mg/kg). Significant test was performed by analysis of variance (ANOVA) and Npar1 way Kruskal-Wallis test. All calculations were performed by PC-SAS system. RESULTS: 1. PZQ was more effective against C. sinensis larvae than against adult worms in the control (P < 0.001), ES Ag (P < 0.01) or crude AW Ag immunization group (P < 0.001). 2. As compared with the control, the worm reduction rate after challenge infection was significantly higher (P < 0.001) in ES Ag immunized group (35.6%) and metacercaria infection group (97.5%) and less in crude AW Ag group (23.4%). The PZQ efficacy was significantly enhanced in ES Ag immunized group. CONCLUSION: The efficacy of PZQ against C. sinensis could be synergically enhanced in rats by inducing host acquired immunity.

Changes of splenocyte IFN-gamma mRNA synthesis in rats infected with Paragonimus westermani.

Changes in the expression level of splenocyte IFN-gamma mRNA of Sprague-Dawley (SD) rats infected with Paragonimus westermani were analyzed by competitive reverse transcription-polymerase chain reaction (RT-PCR) followed by southern blot. The template RNA was extracted from the splenocytes of rats infected with 20 metacercariae of P. westermani. The products of competitive RT-PCR were subjected to southern blot and enhanced chemiluminescence (ECL), and analyzed with a densitometer. In comparison with that of uninfected control rat splenocytes (value of 1), the levels of mRNA expression of IFN-gamma had changed to 0.747 at 1 week post infection (PI), 0.00175 at 2 week PI, 0.0217 at 3 week PI, 0.194 at 4 week PI and then to 0.537 at 5 week PI. The level at 7 week PI had returned to 1.25, comparable with that of uninfected rats. These results show that, when infected with P. westermani, the levels of IFN-gamma mRNA of SD rat splenocytes were remarkably reduced by more than 500 times at 2 week PI and restored to normal level at 7 week PI.

The infestation states and changing patterns of human infecting metacercariae in freshwater fish in Kyongsang-do and Kyonggi-do, Korea.

The infestation rates and abundances of human infecting metacercariae (Clonorchis sinensis, Metagonismus spp., Centrocestus armatus, Echinostoma hortense, Echinochasmus japonicus, Clinostomum complanatum) in freshwater fish were investigated at the three river areas-Taewhagang (river), Hyongsangang (river), Nakdonggang (river) -in Kyongsang-do and at four streams-Yonpungchon, Munsanchon, Kyonganchon, and Konjiamchon-in Kyonggi-do, Korea in 1994-1995. The fish caught at Taewhagang were heavily infested with metacercariae of Clonorchis sinensis and Centrocestus armatus. At Hyongsangang, Zacco platypus and Z. temmincki were moderately infested with metacercariae of C. armatus. Chomanpo, at the basin of Nakdonggang, was still endemic for C. sinensis. In the fish caught at four streams of Kyonggi-do, metacercariae of C. sinensis exhibited the highest infestation rate and intensity out of 6 species of metacercariae. The infestation intensity of C. sinensis metacercariae in fish flesh was markedly different according to each division of flesh. The cause of this difference was conjectured as a result of larval behavior. The metacercariae of C. armatus were found in almost all parts, except scales and fins, of fish. The infestation rates and intensities of C. sinensis and C. armatus metacercariae in Taewhagang greatly increased as compared with those of previous reports. Rhinogobius brunneus and Acanthorhodeus macropterus are newly recorded intermediate hosts of Echinostoma hortense. The reason of large differences from previous data was discussed and the standard method of metacercaria examination was proposed.

Classification and host specificity of Metagonimus spp. from Korean freshwater fish.

Taxonomic problems of Metagonimus spp. in Korea were investigated. Metacercariae of various freshwater fish species -- Plecoglossus altivelis, Carassius auratus, Zacco platypus, Zacco temmincki, Opsariichthys bidens -- were collected from different localities in Korea and experimentally fed to golden hamsters. Observation of recovered adult worms showed that Plecoglossus altivelis was infected with metacercariae of both M. yokogawai and M. takahashii. C. auratus was infected with metacercariae of M. takahashii and Z. platypus, Z. temmincki, O. bidens were infected only with metacercariae of Metagonimus Miyata type. From the inferences about the morphological characteristics, host specificities and occurrence patterns in infected animals, Metagonimus Miyata type is considered to be an independent group.

Gorgoderid trematodes (Digenea: Gorgoderidae) from the urinary bladder of frogs in Korea.

Two species of the family Gorgoderidae (Trematoda: Digenea), Gorgodera japonica Yamaguti, 1936 and Gorgoderina bombinae Yu & Lee, 1983 were collected from the urinary bladder of frogs captured from various localities in Korea. The morphology of each species is described and illustrated. Gorgodera japonica differs from G. cygnoides by having deeply branched vitellaria. Gorgoderina bombinae is considered a valid species.

Demonstration of species-specific and cross reactive components of Paragonimus westermani crude worm antigen by EITB.

Enzyme-linked immunoelectrotransfer blot (EITB) using crude worm antigen of adult Paragonimus westermani was performed for human patients sera to identify the species-specific components. Crude antigen was obtained by homogenizing and centrifuging 24-week old adult worms at 10,000 rpm for 60 minutes in phosphate buffered saline (PBS, pH 7.2) containing phenyl methyl sulfonyl fluoride (PMSF). Gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and blotted electrophoretically onto a sheet of nitrocellulose paper. The sheet was cut into strips and exposed to sera diluted 1: 200 with PBS. SDS-PAGE showed 26 protein bands ranging 229 to 10 kDa. Of them 229, 91, 60, 50, 35-31, 27, 25, 21, 17, 11 and 10 kDa components showed positive reaction with serum antibody of patients with P. westermani. Sera of patients infected with Clonorchis sinensis reacted with 35-31, 19, and 11 kDa bands. Human sera from cysticercosis and diphyllobothriasis cases showed non-specific cross reactions with 229, 35-31, 27, 25 and 17 kDa bands. Protein bands of 91, 60, 21 and 10 kDa showed strong positive reaction without cross reactions with sera from other helminthic infections.

[Analysis of antigenic specificities of Paragonimus westermani developmental stages using immunoblot technique].

Serodiagnosis of parasitic infections is widely used, since parasites or their eggs are not always detected by ordinary methods. The sensitive tests such as ELISA are highly dependent on the purity of antigens used. To solve this problem, many workers have tried to find species-specific components of antigens. The present study was performed to determine the antigenic profile of crude saline extracts of 3, 5, 8 and 12-week old P. westermani worms, which were collected from experimentally infected cats, based on SDS-PAGE and immunoblot technique. The results were as follows: 1. The SDS-PAGE showed at least 30 protein bands ranging from 229 kDa to 10 kDa molecular weight. The protein components of P. westermani changed chronologically during its developmental period. The 229 kDa band was recognized only in 12-week old worms (SEP12). 2. Analysis by ELISA showed a significant increase in antibody levels at 3 weeks in infected cats using crude saline extract antigens (SEP3, SEP5, SEP8, SEP12). 3. By EITB using SEP3 and SEP5, infected cats recognized major protein bands with molecular weight of 60, 35, 28, 25 or 21 kDa at 3-12 weeks of infection, and 3 additional antigens, 19, 13 and 10 kDa, were detected at 8-12 weeks of infections. 4. Using SEP8, 5 antigens, 91, 85, 31, 25 and 21 kDa, were consistently detected by all infected sera tested. In addition, 3 antigens of 19, 13 and 10 kDa were detected at 8-12 weeks of infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical evaluation of the therapeutic efficacy of praziquantel against human cysticercosis.

Thirty-three patients with confirmed cysticercosis, with or without cerebral involvement, were treated with praziquantel at a daily dose of 75 mg/kg tid for 3 to 10 consecutive days and evaluated for tolerance and therapeutic effects. For dermal cysticercosis, a daily dose of 75 mg/kg tid for 3 days is effective. The nodules disappeared in one to 12 months after treatment. Twenty out of 33 patients with cysticercosis had cerebral cysticercosis. Among these 20 cases, 13 had epileptic seizures, 6 had a combination of both epileptic seizures and intracranial hypertension, and one had headache. The efficacy of the treatment of cerebral cysticercosis was assessed by the frequency of convulsions before and after treatment, and by the disappearance or decreased densities of the cystic lesions at brain CT scanning (6 months follow up). A daily dose of 75 mg/kg tid for 7 to 10 days (total 525 to 750 mg/kg) seems to be effective for the treatment of cerebral cysticercosis. The concomitant oral administration of dexamethasone during the course of treatment was effective for preventing and minimizing side effects. Praziquantel was very well tolerated.