Metagonimus yokogawai: a 100-kDa Somatic Antigen Commonly Reacting with Other Trematodes

This study was undertaken to characterize the properties of a 100 kDa somatic antigen from Metagonimus yokogawai. Monoclonal antibodies (mAbs) were produced against this 100 kDa antigen, and their immunoreactivity was assessed by western blot analysis with patients' sera. The mAbs against the 100 kDa antigen commonly reacted with various kinds of trematode antigens, including intestinal (Gymnophalloides seoi), lung (Paragonimus westermani), and liver flukes (Clonorchis sinensis and Fasciola hepatica). However, this mAb showed no cross-reactions with other helminth parasites, including nematodes and cestodes. To determine the topographic distribution of the 100 kDa antigen in worm sections, indirect immunoperoxidase staining was performed. A strong positive reaction was observed in the tegumental and subtegumental layers of adult M. yokogawai and C. sinensis. The results showed that the 100 kDa somatic protein of M. yokogawai is a common antigen which recognizes a target epitope present over the tegumental layer of different trematode species.

Regulation of anti-inflammatory cytokines IL-10 and TGF-beta in mouse dendritic cells through treatment with Clonorchis sinensis crude antigen

Dendritic cells (DCs), which are regarded as the most potent antigen-presenting cells, are involved in innate and adaptive immunity. Upon uptake of pathogens, DCs express cell surface markers and secrete cytokines. In this study, we analyzed production of cytokines and found that interleukin (IL)-10 and transforming growth factor (TGF)-beta production significantly increased in bone marrow-derived DCs and a mouse DC line, DC2.4, after treatment with crude antigen (CA) from liver fluke, Clonorchis sinensis. However, expression patterns of several activation molecules did not change. In addition, following treatment of DC2.4 cells with antigen from the lung fluke, Paragonimus westermani, production of IL-10 and TGF-beta significantly increased compared with groups treated with other parasite antigens, Spirometra erinacei plerocercoid CA and Echinococcus granulosus hydatid cystic fluid. We also found that treatment of DC2.4 cells with C. sinensis CA resulted in rapid and significant phosphorylation of extracellular signal-regulated kinase 1/2, a mitogen-activated protein kinase. Following treatment of DC2.4 cells with C. sinensis CA, treatment with an inhibitor specific to an extracellular signal-regulated kinase inhibited production of IL-10 and TGF-beta. Our results suggest that CA from C. sinensis has a role in the anti-inflammatory function of DC cells by inducing IL-10 and TGF-beta through activation of extracellular signal-regulated kinase 1/2.

A Case of Fascioliasis in the Intrahepatic Duct with Concurrent Clonochiasis / 대한소화기학회지

The main causes of biliary obstruction are stones and cancers. Fascioliasis is a very rare case which causes biliary obstruction. Fascioliasis is a zoonosis caused by Fasciola hepatica which infects herbivores like sheep and cattle. F. hepatica lives in the biliary system or the liver parenchyma of a host. In Korea, the occurrence of this infection in human is very rare and only few cases have been reported. A 32-year-old male presented with upper abdominal pain and jaundice. His laboratory finding revealed elevated liver transaminases. Abdomen CT scan showed mild left intrahepatic bile duct dilatation. On ERCP, adult F. hepatica worms were found and were thus removed. Concurrently, clonorchiasis was diagnosed by stool exam and serologic enzyme-linked immunosorbent assay test. Clonorchiasis was treated with praziquantel. Herein, we report a case of intrahepatic bile duct dilatation due to F. hepatica infection with concurrent Clonorchis sinensis infestation.

Seroprevalence of Tissue Invading Parasitic Infections Diagnosed by ELISA in Korea

Seroprevalence of the IgG antibodies for Clonorchis sinensis, Paragonimus westermani, Taenia solium metacestode (cysticercus), and Spirometra erinacei plerocercoid (sparganum) was measured using enzyme-linked immunosorbent assay (ELISA) in sera of patients in Korea from 1993 to 2006. A total of 74,448 specimens referred nationwide from 121 hospitals revealed an IgG positive rate of 7.6% for the 4 parasites. The IgG positive rate (18.7%) for the 4 parasites in 1993 decreased gradually to 6.6% in 2006. Individual positive rate decreased from 5.2% (1993) to 1.6% (2006) for C. sinensis, from 2.8% (1993) to 1.1% (2006) for P. westermani, from 8.3% (1993) to 2.2% (2006) for cysticercus, and from 2.6% (1993) to 1.6% (2006) for sparganum. The positive rate was highest (21.2%) in the group of patients who ranged in age from 50-59 yr old, and in the group that was referred from the Seoul area (55.9%). In conclusion, our results suggest that tissue invading parasitic infections should always be included in differential diagnosis for patients with eosinophilia associated lesions of the central nervous system, liver, and lungs in Korea.

Changing Patterns of Serum and Bile Antibodies in Re-infected Rats with Clonorchis sinensis

Rats develop strong resistance to re-infection and super-infection by Clonorchis sinensis. The present study investigated the antibodies present in the sera and bile juice of rats that were primary infected and re-infected with C. sinensis. The serum level of specific IgG antibodies, which were elevated 2 wk of the primary infection, peaked at 4 wk and subsequently remained unchanged even during re-infection. The total IgE level in serum increased slowly from 388 ng / ml to 3,426 ng / ml beginning 2 wk after the primary infection, and remained high up to 8 wk but dropped to a normal level (259 ng / ml) after treatment. In resistant re-infected rats, the serum IgE level increased rapidly and peaked within 1 wk, whereas no increase was observed in immunosuppressed rats. The serum level of specific IgA antibodies was elevated beginning 1 wk after infection, and decreased 4 wk after treatment. The total bile IgA level unchanged during the primary infection but increased in treated and re-infected rats. The elevated levels of serum IgE and bile IgA indicate that these immunoglobulins may be correlated with the development of resistance to re-infection by C. sinensis in rats.

Changing Patterns of Serum and Bile Antibodies in Re-infected Rats with Clonorchis sinensis

Rats develop strong resistance to re-infection and super-infection by Clonorchis sinensis. The present study investigated the antibodies present in the sera and bile juice of rats that were primary infected and re-infected with C. sinensis. The serum level of specific IgG antibodies, which were elevated 2 wk of the primary infection, peaked at 4 wk and subsequently remained unchanged even during re-infection. The total IgE level in serum increased slowly from 388 ng / ml to 3,426 ng / ml beginning 2 wk after the primary infection, and remained high up to 8 wk but dropped to a normal level (259 ng / ml) after treatment. In resistant re-infected rats, the serum IgE level increased rapidly and peaked within 1 wk, whereas no increase was observed in immunosuppressed rats. The serum level of specific IgA antibodies was elevated beginning 1 wk after infection, and decreased 4 wk after treatment. The total bile IgA level unchanged during the primary infection but increased in treated and re-infected rats. The elevated levels of serum IgE and bile IgA indicate that these immunoglobulins may be correlated with the development of resistance to re-infection by C. sinensis in rats.

Susceptibility of experimental animals to reinfection with Clonorchis sinensis

The present study observed the resistance to reinfection with Clonorchis sinensis in various experimental animals including mice, guinea pigs, rabbits, and dogs, as well as rats and hamsters. The resistance rates to reinfection in rats, mice, hamsters, guinea pigs, rabbits, and dogs were 79.7%, 58.0%, -12.6%, 54.8%, 62.6%, and 6.0%, respectively. Worms recovered from reinfected rats and mice were immature, and significantly smaller than those from the primarily infected (P < 0.01), whereas those from other animals were fully matured to adults. These findings indicate that the protective response against reinfection with C. sinensis is prominent in rats and mice, and that they may be a good animal model to investigate the mechanism of resistance to reinfection with C. sinensis.

Excretory-secretory antigen is better than crude antigen for the serodiagnosis of clonorchiasis by ELISA

Although stool examination is the standard diagnostic method of clonorchiasis, serodiagnosis by ELISA using crude antigen is now widely used because of its convenience. However, ELISA diagnosis still suffers from cross-reactions, and therefore there is a need to improve the present conventional ELISA. The present study was undertaken to evaluate the diagnostic value of ELISA using excretory-secretory antigen (ESA) instead of crude antigen (CA) of Clonorchis sinensis. The diagnostic sensitivity of ELISA using excretory-secretory antigen was 92.5%, which was higher than that of ELISA using crude Clonorchis sinensis antigen (88.2%). In addition, the specificity of excretory-secretory antigen was found 93.1% while that of crude antigen was 87.8%. In summary, Clonorchis sinensis ESA was found to be a better serodiagnostic antigen than CA for ELISA.

Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini (Opisthorchidae, Trematoda)

The antigenic characterizations and serological reactions of human liver flukes, Clonorchis sinensis and Opisthorchis viverrini, were analyzed by immunoblot. The antigenic profiles of the crude extract of Clonorchis contained major proteins of 8, 26-28, 34-37, 43, and 70 kDa, and those of Opisthorchis 34-37, 43, 70, and 100 kDa. Of these, the 8, 26-28 and 34-37 kDa bands of Clonorchis and the 100 kDa of Opisthorchis were major components of each excretory-secretory antigen. The 8 and 26-28 kDa bands were specific to Clonorchis but the 100 kDa of Opisthorchis cross-reacted with the sera of clonorchiasis, and the 34-37, 70 and 100 kDa bands cross-reacted with sera of other helminthiases. The frequency and intensity of the immunoblot reactions were positively correlated with the intensity of the liver fluke infection.

Identification of IgE-reacting Clonorchis sinensis antigens

Clonorchis sinensis is a liver fluke and it is the most prevalent human parasite in Korea at present. The parasite infection induces immune responses, characteristically an increased production of parasite-specific IgE in the host. Major IgE-reacting C. sinensis antigens in infected humans have been protein bands with MWs of 15, 28, 37, 45, 51, 56, 62, 66, 74, 97 and 160 KD identified by immunoblot analysis. Individual variations of the IgE binding pattern to C. sinensis antigens have also been documented. Using immune BALB/c mouse sera, IgE-reacting protein bands have been visualized with MWs of 28, 74, 86, 160 and several > 200 KD. One of the most strongly reacted C. sinensis antigenic proteins with a molecular weight of 28 KD was purified by gel filtration and preparative electrophoresis. Using a monoclonal antibody produced against the antigenic protein, the protein was localized in the parasite's intestine, and also found to be contained in excretory-secretory antigens.

Antibodies in serum of patients with clonorchiasis before and after treatment.

Sera of 31 patients infected with Clonorchis sinensis were examined using fraction 1 antigen by ELISA during a one-year observation. The results of ELISA with Igs, IgG and IgA demonstrated high sensitivity (100%, 100% and 90%) and specificity (100%, 100% and 87%). Sera specific Igs and IgG were significantly decreased in the 3rd month after treatment with praziquantel (25mg/kg body weight in one dose), and IgA was significantly decreased in the 1st month (paired t-test, p < 0.05). No eggs were found in the stool after treatment. Detection of sera specific Igs, IgG and IgA by ELISA was combined with stool examination to evaluate the effect of praziquantel and the completeness of the cure.

Detection of specific serum IgE in clonorchiasis cases and analysis of Clonorchis sinensis allergens

Specific serum IgE levels of Clonorchis sinensis in infected humans were measured by avidin-biotin ELISA, and allergens from C. sinensis were identified by immunoblot and autoradiography. Then, allergens fractionated by Sephadex G-200 gel filtration were analyzed, and cross-reactive allergenic components of C. sinensis reacted with paragonimiasis sera were revealed. Fourteen out of 15 C. sinensis egg-positives were found to be serum IgE positive (absorbance > 0.27). Of 14 IgE-reacting allergen bands visualized, major allergens of 66, 61.5, 45, 37, 28.5, 23.5 and 15.5 KD were recognized by more than 50% of the sera of infected humans. The considerable individual variations of IgE immune responses to C. sinensis allergenic components were also noticed. C. sinensis extract was separated into 5 fractions by Sephadex G-200 gel filtration. Seventy-four KD allergen was recognized in the first fraction, 50, 45, 37, 29.5 and 28.5 KD in the third, and 15.5 KD in the fourth. Cross-reactive allergens with sera of paragonimiasis cases were identified as 66, 45, 28.5, 13 and 7.5 KD.

Diagnosis of clonorchiasis by ELISA-inhibition test using a Clonorchis sinensis specific monoclonal antibody.

The ELISA-inhibition test using Clonorchis sinensis specific monoclonal antibody (CsHyb 0605-23) for diagnosis of clonorchiasis was carried out. It demonstrated sensitivity and high specificity in comparison with the conventional ELISA.

Counterimmunoelectrophoresis test on human Clonorchis sinensis infection.

A counterimmunoelectrophoresis test was used to detect antibodies against the adult worm antigen of Clonorchis sinensis in sera from 70 clonorchiasis patients, 20 uninfected healthy persons and 7 patients infected with other helminths. A constant voltage of 10 V/cm and a running time of 30 minutes was chosen in carrying out detection. Antibody titers of 1, 1:2 and 1:4 were obtained from 35, 21 and 14 clonorchiasis patients, respectively. Significant correlation was observed between worm burden in patients and antibody titer, the higher the antibody titer in patients, the more eggs per gram feces in their stool. Although cross reaction was observed with toxocariasis and angiostrongyliasis in this study, high (100%) sensitivity made it possible to screen the subjects in endemic areas to shorten the survey period.

Immunodiagnosis of clonorchiasis by enzyme-linked immunosorbent assay.

The present study applied the indirect enzyme-linked immunosorbent assay (ELISA) technique in the immunodiagnosis of clonorchiasis. Antigen used in this study was extracted from adult worms of Clonorchis sinensis obtained from cats. 132 patients with clonorchiasis, 100 healthy persons and 14 patients with other parasitic infections were studied. Mean O.D. ratio with standard deviation of clonorchiasis was 1.41 +/- 0.21 with 0.95 +/- 0.13 of healthy persons. Results revealed 90.2% to 95.5% of sensitivity and 84% to 99% specificity dependent on the two cut off values of O.D. ratio, i.e. 1.10 and 1.20. Antibody titers derived from O.D. ratio highly correlated with direct titration (Y = 0.0303 +/- 1.1766 X, r = 0.8945). Cross reactions of other parasite infections to clonorchiasis were observed in patients with angiostrongyliasis and schistosomiasis.

Anti-P1, allohemagglutinins associated with Clonorchis sinensis and opisthorchis viverrini infections in patients from Southeast Asia.

An automated assay of anti-P1 allohemagglutinins has been carried out on sera of 61 individuals from Southeast Asia : 28 with clonorchiasis, 18 with opisthorchiasis and 15 control subjects. Anti-P1 activity was detected in 61% of the opisthorchiasis sera, 57% of the clonorchiasis sera and in 26.6% of the control subjects. Their concentration, in the sera, was low in control subjects and exceptionally high in clonorchiasis and opisthorchiasis (up to 13 and 22 times the maximum concentration of the control subjects, respectively). In all cases the anti-P1 antibodies were of IgM class. The results suggested that Clonorchis and Opisthorchis were responsible for immunization of the patients, with P1 alloantigen.