In order to obtain the most specific and sensitive antigen from crude antigens of Fasciola hepatica for the immunodiagnosis of bovine fascioliasis by the enzyme linked immunosorbent assay(ELISA), phosphate buffered saline extract of F. hepatica was prepared. The crude extract was fractionated into 7 antigens using Sephadex G-100 column chromatography. Seven fractionated antigens were applied to ELISA, precipitation test and intradermal test, respectively. Results obtained are as follows: The specificity (95 per cent confidence interval in negative sera of bovine fascioliasis ; Mean+2 x SD of absorbance ) of the first (MW>150,000) and the second antigens (MW 120,000) were 93.7 per cent, but those of others including crude antigen showed 100 per cen.t. The sensitivity (positive sera of bovine fascioliasis having higher values with compared to the criterion) of the first, the sixth (MW 16,000) and the seventh antigen (MW<5,000) were 91.6 per cent, 87.5 per cent and 0 per cent, respectively, but those of others showed all 100 per cent. The absorbance by ELISA using the fifth antigen (MW 26,000) was 8.43-folds higher in the positive sera than that in the negative sera. This could be used as one of the most specific antigens for the immunodiagnosis of bovine fascioliasis. In Ouchterlony test, precipitin lines were not found in the sera naturally infected with F. hepatica, but some were found in the sera of rabbits immunized with the crude antigens. The numbers of precipitin lines in the sera of rabbits were different in the different fractionated antigens. They were 6 in the crude, 2 in the second and the third antigens, 1 in the forth, the fifth and the sixth antigens and absent in the seventh antigen, respectively. The wheal size for the bovine infected with F. hepatica was 2.46+-0.15 cm in the intradermal test antigen (saline extract of F. hepatica) supplied by the Veterinary Research Institute, Rural Development Administration, Korea. The wheal size of the first, the second and the third antigens were larger than that of intradermal test antigen, whereas those of the forth, the fifth, the sixth and the seventh antigens showed smaller than that of the intradermal test antigen. The results suggest that the fifth antigen may be specific antigen for the immunodiagnosis of bovine fascioliasis.
parasitology-helminth-trematoda ; immunology ; enzyme-linke immunosorbent assay ; diagnosis ; fascioliasis ; Fasciola hepatica

In an attempt to investigate the specific antigenic substance of Fasciola hepatica, Ouchterlony tests and immunoelectrophoretic analyses were carried out. Crude Fasciola antigen was prepared and fractionated by Sephadex G-200 column to Antigen I, II and III according to protein content. Crude antigens of Paragonimus westermani, Clonorchis sinensis and Paramphistomum sp. were also prepared for control and absorption study. Antiserum was prepared by injecting 0.5 ml of crude Fasciola antigen with same amount of complete Freund's adjuvant in rabbits, 10 times at an interval of l week. The results obtained in this study were as follows: Crude Fasciola antigen reacted with antiserum with 9 precipitin bands by Ouchterlony test and with 11 bands by immunoelectrophoresis. Cross reaction was observed between Paragonimus, Clonorchis and Paramphistomum antigens and anti-Fasciola rabbit serum respectively. By Ouchterlony test, 3-4 cross reacting bands were found. Anti-Fasciola sera which were absorbed with respective Paragonimus, Clonorchis and Paramphistomum antigens, reacted with Fasciola crude antigen. Ouchterlony test gave 5-6 precipitin bands. Further reaction between Fasciola antigen and antiserum absorbed with the above 3 antigens concomitantly gave 5 precipitin bands by Ouchterlony test and 7 bands by immunoelectrophoretic analyses. Fractionated Fasciola antigens (Antigens I, II and III) reacted with anti-Fasciola rabbit serum in immunoelectrophoresis. Antigen I, II and III gave 2, 3 and 5 precipitin bands respectively. Anti-Fasciola rabbit serum which was absorbed with 3 trematodes antigens gave, by immunoelectrophoresis, 1 band with Antigen I, 2 bands with Antigen II and III of Fasciola hepatica. From the above results, it is concluded that Fasciola hepatica possessed the specific antigenic substance not cross-reacted with other trematodes.
parasitology-helminth-trematoda ; Fasciola hepatica ; Paragonimus westermani ; Clonorchis sinensis ; Paramphistomum sp. ; antigen ; immunology ; electrophoresis

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.
Animals ; Antibodies, Helminth/immunology ; Antibodies, Monoclonal/*immunology ; Antigens, Helminth/*immunology ; Clonorchis sinensis/immunology ; Cross Reactions/immunology ; Fasciola hepatica/immunology ; Female ; Helminth Proteins/*immunology ; Heterophyidae/*immunology ; Immunologic Tests ; Mice ; Mice, Inbred BALB C ; Paragonimus westermani/immunology ; Trematode Infections/*diagnosis/immunology

In an attempt to investigate the antigen-antibody relations and the value of immunodiagnosis for several helminths, Ouchterlony tests and immunoelectrophoreses were carried out. Taenia saginata, Cysticercus sp. of cestodes, Clonorchis sinensis, Fasciola hepatica and Paramphistomum sp. of trematodes,and Ascaris suum of nematodes were used as antigens. On the other hand, antisera were obtained by injecting 0.5 ml each of the above antigens and the same amount of complete Freund's adjuvant into rabbits ten times at an interval of one week. The result obtained in this study are as follows: A larger number of precipitin arcs were demonstrated in homologous antigen-antibody reactions than in heterologous antigen-antibody reactions both in Ouchterlony tests and immunoelectrophoreses. Gross reactions were observed between the different species of the same class, but no cross reactions were noticed when the classes were different with one or two exceptions, such as between T. saginata, F. hepatica and A. suum. In A. suum, the difference between the male and female was more distinct in Ouchterlony test and immunoelectrophoresis than in the examination of organs such as genital organ and coeliac fluid. Immunoelectrophoresis revealed specific arcs and higher sensitive reaction than Ouchterlony test, and was considered to be a more valuable method for identifing species and immunological diagnosis.
parasitology-helminth-trematoda ; cestoda ; nematoda ; immunoelectrophoresis ; Taenia saginata ; Cysticercus ; Clonorchis sinensis ; Fasciola hepatica ; Paramphistomum sp. ; Ascaris suum ; antigen ; immunology

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG1, and IgG2 (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-gamma, and TNF-alpha, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-beta, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.
Animals ; Antibodies, Helminth/immunology ; Cysteine Proteases/administration & dosage/*immunology/isolation & purification ; Cytokines/immunology ; Fasciola/chemistry/*enzymology/immunology ; Fasciola hepatica/immunology/physiology ; Fascioliasis/immunology/parasitology/*prevention & control ; Female ; Helminth Proteins/administration & dosage/*immunology/isolation & purification ; Humans ; Male ; Protective Agents/*administration & dosage/isolation & purification ; Sheep ; Vaccines/immunology

Although schistosomicidal drugs and other control measures exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. In this study, native fatty acid binding protein (FABP) from Fasciola gigantica was purified from the adult worm's crude extract by saturation with ammonium sulphate followed by separation on DEAE-Sephadex A-50 anion exchange chromatography and gel filtration using Sephacryl HR-100, respectively. CD1 mice were immunized with the purified, native F. gigantica FABP in Freund's adjuvant and challenged subcutaneously with 120 Schistosoma mansoni cercariae. Immunization of CD1 mice with F. gigantica FABP has induced heterologous protection against S. mansoni, evidenced by the significant reduction in mean worm burden (72.3%), liver and intestinal egg counts (81.3% and 80.8%, respectively), and hepatic granuloma counts (42%). Also, it elicited mixed IgG1/IgG2b immune responses with predominant IgG1 isotype, suggesting that native F. gigantica FABP is mediated by a mixed Th1/Th2 response. However, it failed to induce any significant differences in the oogram pattern or in the mean granuloma diameter. This indicated that native F. gigantica FABP could be a promising vaccine candidate against S. mansoni infection.
Animals ; Antibodies, Helminth/immunology ; Fasciola/*chemistry ; Fatty Acid-Binding Proteins/*administration & dosage/immunology/isolation & purification ; Female ; Helminth Proteins/*administration & dosage/immunology/isolation & purification ; Humans ; Immunization ; Mice ; Mice, Inbred Strains ; Schistosoma mansoni/immunology/*physiology ; Schistosomiasis mansoni/immunology/parasitology/*prevention & control

This study investigated the presence of cytokines interferon (IFN)-gamma, interleukins (IL) -6 and -8 in serum of cattle and buffaloes infected with Fasciola gigantica from one to 16 weeks post-infection to determine their T cell response during infection. The concentration of these cytokines was determined by sandwich enzyme-linked immunosorbent assay (ELISA). No IFN-gamma was detected in these animals while IL-6 was elevated from one to 16 weeks postinfection. Levels of IL-8 were also elevated in infected buffaloes from one to 16 weeks post-infection. A predominantly T helper (Th) 2 response which started early in the infection was apparently present in cattle and buffaloes in this study which was characterised by IL-6. IL-8 production could be another mechanism of immune response in buffaloes during infection with F. gigantica.
Animals ; Buffaloes/blood/immunology/*parasitology ; Cattle ; Cattle Diseases/*immunology/*parasitology ; Cytokines/*blood/immunology ; Enzyme-Linked Immunosorbent Assay/veterinary ; Fasciola/*immunology ; Fascioliasis/immunology/parasitology/*veterinary ; Interferon Type II/blood ; Interleukin-6/blood ; Interleukin-8/blood ; Random Allocation

This study was designed to detect and evaluate an antigenicity of low molecular weight proteins of Fasciola hepatica in fascioliasis. Low molecular weight protein of F. hepatica was purified by ammonium sulfate precipitation and Sephacryl S-100 HR gel filtration. The protein obtained was estimated to be 8 kDa on 7.5-15% gradient sodium dodecyl sulfate gel electrophoresis. Immunoblotting studies showed that the 8 kDa protein reacted with human fascioliasis sera, but not other trematodiasis sera. This result suggests that the 8 kDa protein of F. hepatica is one of diagnostic antigens in human fascioliasis without cross-reaction with other human trematodiasis.
Animals ; Antigens, Helminth/*isolation & purification ; Electrophoresis, Polyacrylamide Gel ; Fasciola hepatica/immunology/*isolation & purification ; Fascioliasis/blood/*parasitology ; Helminth Proteins/*isolation & purification ; Human ; Immunoblotting

In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19+ B cells was observed as early as week 1 post-infection while CD4+/CD8+ T cells were down-regulated. Accumulation of Mac1+ cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-alpha mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1beta expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-beta were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-beta and IL-4 during the early stages of infection.
Animals ; B-Lymphocytes/immunology ; CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; Down-Regulation ; Fasciola hepatica/*immunology ; Fascioliasis/*immunology ; Immunophenotyping ; Immunosuppression ; Interleukin-4/blood/genetics/*immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Spleen/immunology ; Transforming Growth Factor beta/blood/genetics/*immunology

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.
Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/*diagnostic use ; Diagnostic Tests, Routine/*methods ; Egypt ; Fasciola/immunology/*isolation & purification ; Fascioliasis/*diagnosis/parasitology ; Humans ; Immunoblotting/*methods ; Parasitology/*methods ; Predictive Value of Tests ; Sensitivity and Specificity