Isolation of Fasciola hepatica tegument antigens

Fasciola hepatica tegument antigens were isolated from intact worms in the cold by using Nonidet P-40. Proof of the tegumental nature of the antigens was shown by the peroxidase-antiperoxidase immunocytochemical technique at the light microscope level. The potential of F. hepatica tegument antigens for the immunodiagnosis of rabbit and human fascioliasis was shown by Ouchterlony immunodiffusion, although cross-reactivity was evident in one of six serum samples from patients infected with Schistosoma mansoni. A genus-specific Fasciola antigen was found in F. hepatica tegument. Finally, F. hepatica tegument contained antigens which protected mice from challenge infection with S. mansoni.     

Mapping of B-cell epitopes on a novel 11.5-kilodalton Fasciola hepatica-Schistosoma mansoni cross-reactive antigen belonging to a member of the F. hepatica saposin-like protein family

FhSAP-2 is a novel member of the Fasciola hepatica saposin-like protein family that induces protection in rabbits against a challenge infection. We investigated the presence of lineal B-cell epitopes within this protein using a set of overlapping synthetic peptides. Peptides were tested in enzyme-linked immunosorbent assays against sera from rabbits infected with F. hepatica. Two dominant epitopes were identified, which were also highly reactive with sera from mice infected with Schistosoma mansoni. These peptides may be suitable to incorporate into a polyepitope-based vaccine formulation against F. hepatica.     

Rapid competitive enzyme-linked immunosorbent assay using a monoclonal antibody reacting with a 15-kilodalton tegumental antigen of Schistosoma mansoni for serodiagnosis of schistosomiasis.

A competitive enzyme-linked immunosorbent assay (CELISA) for antibody detection was developed by using a monoclonal antibody which reacts with a 15-kDa tegumental antigen of the adult worm of Schistosoma mansoni. This monoclonal antibody was not able to react with antigens of Schistosoma japonicum or Schistosoma haematobium in enzyme-linked immunoelectrotransfer blot (EITB) and indirect immunofluorescence tests. The assay was performed in a period of 1 h using an adult worm crude extract antigen. To evaluate the CELISA, a total of 73 serum samples was analyzed: 35 were from S. mansoni-infected patients, 23 were from individuals with parasitic infections other than schistosomiasis, and 14 were from healthy individuals. All serum samples from healthy individuals and from patients infected with other parasites were negative, as were two (6%) samples from patients infected with S. mansoni. EITB analysis showed that 32 of 33 CELISA-positive samples were positive in the EITB but with different patterns of reactivity. A 15-kDa protein reacted with 60% of serum samples, and a 60-kDa protein showed the highest level of reactivity (85%). The two samples from patients infected with S. mansoni that were negative in the CELISA reacted with 70-, 60-, 50-, 47-, and 38-kDa proteins. One sample, positive in CELISA, did not react with proteins of the antigenic extract.     

Application and assessment of a dipstick assay in the diagnosis of hydatidosis and trichinosis

The aim of the present work was to apply and evaluate a dipstick assay for the serodiagnosis of human hydatidosis as well as human and experimental trichinosis using camel hydatid cyst fluid (HCF) and Trichinella spiralis muscle larval (TSML) antigens, respectively, and compare this to enzyme-linked immunoelectrotransfer blot (EITB) and Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA). Sera samples were collected from patients with confirmed hydatidosis and trichinosis and with other parasitic diseases as well as from normal healthy individuals. Also, sera samples were collected from mice experimentally infected with T. spiralis which were sacrificed at different time points post-infection (PI). HCF and TSML antigens were used in EITB after separation and characterization of their antigenic components using 5–22.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis under non-reducing condition. For the diagnosis of hydatidosis, the sensitivity, specificity and diagnostic accuracy of the dipstick assay and EITB were 100, 91.4 and 95.1% while those of FAST-ELISA were 96.2, 100 and 98.4%, respectively. For the diagnosis of human trichinosis, the sensitivity, specificity and diagnostic accuracy of the dipstick assay and EITB were 100% while those of FAST-ELISA were 85.7%. FAST-ELISA proved to be more sensitive in the early diagnosis of experimental T. spiralis infection (100% sensitivity from the second week PI) than the dipstick and EITB (100% sensitivity from the third week PI). All tests retained their sensitivity till the 12th week PI. Since the dipstick assay is extremely easy to perform with a visually interpreted result within 15 min, in addition to being both sensitive and specific, the test could be an acceptable alternative for use in clinical laboratories lacking specialized equipment and the technological expertise needed for EITB and FAST-ELISA.     

Detection of circulating excretory secretory antigens in human fascioliasis by sandwich enzyme-linked immunosorbent assay.

We developed a sandwich enzyme-linked immunosorbent assay to detect circulating parasite antigen in humans with fascioliasis. The assay uses antibodies against Fasciola hepatica excretory secretory (ES) antigens. A monoclonal antibody was used to capture circulating ES antigens, and a human polyclonal antibody peroxidase conjugate was used to identify circulating ES antigens. Optimal dilutions of all reagents were determined by block titration. The antigen concentration in sera from patients was estimated by comparing the optical density at 492 nm of test sera with a standard curve. All of the serum samples from 25 patients with parasitological evidence of fascioliasis had a detectable antigen concentration (more than 10 ng/ml). None of the serum samples from 80 patients infected with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay.     

Immunoprecipitation of in vitro translation products of Fasciola hepatica RNA

Incubation of in vitro translation products of RNA isolated from Fasciola hepatica with immune rat serum and sera from infected sheep resulted in the immunoprecipitation of a number of polypeptides, most of which had similar electrophoretic mobilities. Although only one of these (a 30 kDa species) correlated precisely in mobility with that of biosynthetically labelled excretory-secretory (ES) antigens, this polypeptide (along with several others) was also immunoprecipitated by sheep sera raised against authentic ES antigens. This verified that the isolated RNA contained mRNA species encoding ES antigens of the parasite. In addition, immune rat serum immunoprecipitated a 64 kDa polypeptide which was not immunoprecipitated by infected sheep serum or anti-ES antigen sheep serum. The possible significance of this finding in terms of the ability of rats to develop resistance to fascioliasis in contrast to sheep which apparently cannot is discussed.     

Enzyme-linked immunoelectrotransfer blot test for diagnosis of human hydatid disease.

Sera from 71 patients with surgically confirmed hydatid disease (which is caused by Echinococcus granulosus) were studied by an enzyme-linked immunoelectrotransfer blot (EITB) assay. Sera from patients either with other cestode infections or with another illness were used as controls. Results of the EITB test for hydatidosis were compared with those of the double-diffusion (DD5) test and an enzyme-linked immunosorbent assay (ELISA). In the EITB assay with bovine lyophilized hydatid fluid, three antigen bands of 8, 16, and 21 kDa were diagnostically important. The sensitivity of the assay by using these antigen bands was 80% for hepatic cysts, 56% for pulmonary cysts, and 56% for cysts located in multiple organs. In sera from controls, the specificity of the EITB assay was 100%. Cross-reactions to the 8-, 16-, and 21-kDa bands occurred, respectively, in 12, 4, and 4% of sera from patients with cysticercosis. No cross-reactions were noted in patients infected with Hymenolepis nana. The ELISA in which swine hydatid fluid was used as the antigen was as sensitive as the EITB test but was less specific (80%) and frequently cross-reacted with sera from patients with other cestode infections. The sensitivity of the DD5 test, which uses sheep hydatid fluid, was low (47%) , but its specificity was as high as that of the EITB assay. However, in patients with cysticercosis, cross-reactions were observed in 23% of sera tested. Despite the higher sensitivity found with the EITB assay, 23% (n = 5) of the serum samples that were positive by the DD5 test were not detected by the EITB assay. The EITB assay offers greater sensitivity and specificity than do the ELISA and the DD5 test. The highest proportion of hydatid cases is detected when the EITB and DD5 tests are run simultaneously.     

Translation of Schistosoma mansoni antigens in Xenopus oocytes microinjected with mRNA from adult worms

Oocytes from Xenopus laevis microinjected with RNA isolated from Schistosoma mansoni adult worms translated antigens recognized by sera from infected rats, humans, and from immunized rabbits. The pattern of immunoprecipitated proteins analysed by SDS-polyacrylamide gel electrophoresis was species specific in rats. Serum from infected Fischer rats recognized antigens of 20, 27 and several bands in the 50-60 kDa range whereas serum from infected Brown Norway rats also immunoprecipitated major bands at 29, 43 and 100 kDa. Human infection sera gave a very variable pattern of immunoprecipitation not apparently dependent on the patients' age. At least 20 different antigenic species could be identified ranging from 14 to 150 kDa. Some S. mansoni antigenic proteins could be isolated from the membrane fraction of the oocytes whereas notably the 29 kDa band was present mainly in the soluble fraction. N-Glycosylation of S. mansoni antigens occurred as evidenced by the effects of tunicamycin treatment and concanavalin A binding. A multiple series of bands between 50 and 60 kDa, present in the membrane fraction, were glycosylated and secreted from the oocytes. Monoclonal antibodies to larval stage surface antigens failed to immunoprecipitate oocyte translation products, but sera absorbed with live schistosomula identified at least three putative surface antigens of 100, 43 and 29 kDa. However, the 29 kDa molecule was neither synthesized into membranes, nor secreted from oocytes.     

Characterization and differential expression of a ferritin protein from Fasciola hepatica

Ferritins are proteins that play a central role in maintaining intracellular iron balance. A cDNA clone of Fasciola hepatica (687 bp long) encoding a putative 228-amino acid polypeptide (FhFtn-1) homologous with ferritins of vertebrates and invertebrates was identified. FhFtn-1 contains a conserved motif of the ferroxidase center typical of vertebrate ferritins. Phylogenetic tree analysis showed that FhFtn-1 clusters with two ferritins of Paragonimus westermani, which suggests a common ancestry for the ferritins of these two trematodes. Recombinant FhFtn-1 protein expressed and purified from an Escherichia coli system showed iron-uptake ability. Moreover, FhFtn-1 showed strong reactivity with sera from rabbits infected with F. hepatica for 2-12 weeks, which suggests that this protein could be a potential antigen for immunodiagnosis of fascioliasis. qPCR analysis demonstrated that FhFtn-1-mRNA is expressed at significantly higher levels in adults and unembryonated eggs than in juveniles or miracidia. These results represent the first characterization of a ferritin protein from the liver fluke F. hepatica.     

Development and optimization of the FAST-ELISA for detecting antibodies to Schistosoma mansoni

The Falcon assay screening test (F.A.S.T.) system was used to develop a rapid, sensitive, and quantitative kinetic-based enzyme-linked immunosorbent assay (k-ELISA) for detecting antibodies against Schistosoma mansoni adult microsomal antigens (MAMAs). The FAST-ELISA uses polystyrene beads on sticks molded to the lid of a microtitration plate. The beads are coated with antigen. Reagents and sera are placed in microtitration plates and the beads exposed to reagents by immersion. The exposure time required for a single dilution of serum or other antibody source, conjugate, and substrate is 5 min each. Excluding preparation time, two plates can easily be assayed in 30 min. The optima for assay conditions, reproducibility, quantitative linearity, and sensitivity are delineated. A battery of sera from patients with both homologous and heterologous infections was tested, and a dilution series of a standard reference serum pool was included with each test. Results were expressed in number of units as calibrated against the standard reference sera pool. Antigen-coated bead storage studies were performed with untreated and three chemically treated antigens. The storage stability of MAMA, ability to perform the assay with minimal equipment, sensitivity, short assay time, and ease of operation make the FAST-ELISA ideal for field studies.     

Sandwich enzyme-linked immunosorbent assay for detection of excretory secretory antigens in humans with fascioliasis.

A sandwich enzyme-linked immunosorbent assay has been developed for the detection of Fasciola hepatica excretory secretory (ES) antigens in stool specimens of infected humans. The assay uses antibodies against F. hepatica ES antigens. A monoclonal antibody (ES78, mouse immunoglobulin G2a) was used to capture ES antigens, and a rabbit polyclonal antibody, peroxidase conjugate, was used to identify ES antigens. Thirteen of 14 patients with parasitological evidence of fascioliasis had a detectable concentration of ES antigens (more than 15 ng/ml). None of the stool specimens from controls and from patients with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay. When the 14 patients were retested 2 months after treatment, all of the specimens from the 11 parasitologically cured patients were negative by the antigen detection assay while the specimens from the 3 patients with persisting F. hepatica eggs in their stools remained positive.     

Immunoaffinity fractionation of Schistosoma mansoni worm antigens using human antibodies and its application for serodiagnosis.

A crude Schistosoma mansoni soluble worm antigen preparation (SWAP) was fractionated using an immunoaffinity column consisting of specific human anti-SWAP antibodies obtained from chronic S. mansoni-infected human sera and bound to CNBr-activated Sepharose 4B. The chromatographic separation resulted in three fractions: the unbound material (FW), and the eluted antigens with glycine-HCl (F1) and glycine-HCl-NaCl (F2). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the purified antigens F1 and F2 consisted of several bands when stained with Coomassie blue and silver stain, with molecular weights between 20 X 10(3) and 200 X 10(3). The F1 and F2 fractions in addition to FW and SWAP were used in an enzyme-linked immunosorbent assay (ELISA) to measure antibody levels in sera from schistosomiasis patients. Each individual serum assessed with the purified F2 antigen gave 100% positivity and three to four times higher optical density in comparison to SWAP with only 88% positivity. No detectable cross-reactive antibodies against F2 were found when a limited number of sera from filariasis, fascioliasis and trichinellosis patients were screened. Furthermore, F2 was also used and found to be more sensitive generally in detecting anti-adult worm antibodies than SWAP in recently schistosomiasis-infected persons. Thus, F2 appears to be a highly sensitive and specific reagent for the serodiagnosis of schistosomiasis infection.     

Fasciola hepatica alters coagulation parameters in sheep plasma in vivo and in vitro

The blood-sucking activities of the liver fluke, Fasciola hepatica, are likely to cause alterations in coagulation during the course of infection; and the effect of F. hepatica on various coagulation parameters was studied during the course of acute and chronic fasciolosis of sheep over a period of 17 weeks. Whole blood and plasma samples from infected sheep (with 800 metacercariae each) and uninfected controls were collected weekly until 17 weeks post-infection (w.p.i.) and the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) were determined. Additionally, adult F. hepatica were recovered from bile ducts, incubated for the production of excretory/secretory products (ESP) or homogenised and the effect of fluke products on APTT, PT and TT was determined. Anaemia was evident in infected sheep from 8 w.p.i. until 17 w.p.i. Plasma APTT was accelerated during 8, 9, 12, 14, 16 and 17 w.p.i., while PT was prolonged at 8-11 w.p.i. and TT at 10, 14 and 17 w.p.i. Addition of worm ESP or homogenate to plasma resulted in an enhancement of the intrinsic pathway (APTT) together with a prolongation of the extrinsic and common pathways (PT, TT) of coagulation. It was concluded that F. hepatica contains and releases substances that may contribute to coagulation changes in vivo. Further characterisation of the active substance(s) in vitro revealed heat inactivation, a size >30 kDa and inhibition by the proteinase inhibitors Complete and EDTA for the APTT-accelerating substance(s). The TT-deceleration, in contrast, was increased after heating.     

Characterization of Chlamydia pneumoniae species-specific proteins immunodominant in humans.

Proteins of Chlamydia pneumoniae immunodominant in humans were characterized with the sera of 13 patients who were not likely to have been exposed to C. trachomatis or C. psittaci. The serological responses among these patients were similar on a qualitative basis, but some differences were found quantitatively. However, the serological responses of the patients who were infected with C. pneumoniae differed markedly from those of two patients who were infected with C. trachomatis and two who were infected with C. psittaci and those of mice that were transtracheally infected with C. pneumoniae. Among proteins immunodominant in the patients who were infected with C. pneumoniae, a 40-kDa major outer membrane protein was genus specific and 53-, 46-, and 43-kDa proteins were species specific in their reactions with the majority of the human sera used. A few sera reacted strongly with a 73-kDa protein genus specifically. Some proteins with weak immunogenicity exhibited species specificity. An antigenic analysis with human sera and murine monoclonal antibodies against the 53-kDa protein showed that hte antigenicities were strictly conserved among the seven strains of C. pneumoniae tested. The genus-specific 73-kDa protein was solubilized with octylglucoside. All of the species-specific immunodominant proteins were solubilized with sodium dodecyl sulfate, but the genus-specific major outer membrane protein was not. These results suggest that a serological diagnosis of C. pneumoniae infection could be achieved species specifically by comparison of the serum responses to sodium dodecyl sulfate- and octylglucoside-soluble fractions.     

Identification of GTP-binding proteins in Fasciola hepatica and Schistosoma mansoni by immunoblotting

Both the liver fluke Fasciola hepatica and the blood fluke Schistosoma mansoni have GTP-binding proteins which are part of the trans-membrane signalling system. These proteins require GTP in order to interact with the catalytic subunit of adenylate cyclase. The technique of immunoblotting was used in order to distinguish the GTP-binding proteins Gs, Gi, and Go. Immunoblotting was carried out using antisera prepared against peptides deduced from bovine cDNA clones specific for alpha or beta subunits of known G proteins. A 41-kDa Gs alpha has been identified in S. mansoni and a 42.5-kDa Gs alpha in F. hepatica. A 41-kDa Go alpha was found in both parasites. A 36-kDa G beta was identified in both parasites using antiserum made against bovine transducin.     

Detection of antibodies against ovum antigens in Schistosoma mansoni bilharziasis

Fifty-five sera from bilharziasis patients from West Indies and thirty-five control sera (from normal subjects and patients with hydatidosis and fascioliasis) were examined by ELISA for antibodies reacting with S. mansoni soluble egg antigen MSA1 . MSA1 antigen was prepared according to Pelley and coupled to isothiocyanate substituted plastic discs. Sensitivity and specificity of reaction were good. Moreover, in experimental infection in mice, sera of mice cured with praziquantel became negative. The sera were also examined by a whole egg antigen in counter immunoelectrophoresis. The results of the two different techniques correspond well although ELISA using purified antigen prove to be more specific and sensitive.     

Immunodetection of Fasciola gigantica Circulating Antigen in Sera of Infected Individuals for Laboratory Diagnosis of Human Fascioliasis

Currently, the laboratory diagnosis of human fascioliasis is based on the parasitological examination of parasite eggs in stool specimens and serological detection of specific antibodies in serum samples, which are often unreliable diagnostic approaches. Ideally, a sensitive and specific diagnostic test for Fasciola infection should be based on the detection of circulating Fasciola antigen, which implies active infection. Here, a 27-kDa-molecular-mass antigen was identified in a Fasciola gigantica adult worm antigen preparation, excretory-secretory products, and sera from F. gigantica-infected individuals, and it was not detected in antigenic extracts of other parasites and sera from noninfected individuals. The target antigen was isolated and partially characterized as a protein. Immunoperoxidase staining located the target epitope within teguments and guts of F. gigantica adult worms. The performance characteristics of a newly developed enzyme-linked immunosorbent assay (ELISA) based on F. gigantica circulating antigen detection in serum (FgCA-27 ELISA) were investigated using sera of 120 parasitologically diagnosed F. gigantica-infected individuals and 80 noninfected individuals. The area under the receiving operating characteristic (ROC) curve (AUC) for ELISA was significantly high (AUC = 0.961, P < 0.0001) for discriminating Fasciola-infected and noninfected individuals. The developed assay showed high degrees of sensitivity, specificity, and efficiency (>93%), and a significant correlation (r = 0.715, P < 0.0001) between antigen level and parasite egg count was shown. In conclusion, a 27-kDa Fasciola antigen was identified in sera of F. gigantica-infected individuals. A highly sensitive and specific Fasciola antigen detection assay, FgCA-27 ELISA, was developed for laboratory diagnosis of human fascioliasis.     

Development and evaluation of a Western blot kit for diagnosis of human trichinellosis

We evaluated industrially prepared Western blot strips designed to avoid the cross-reactions observed with indirect immunofluorescence and enzyme-linked immunosorbent assays used for the serodiagnosis of trichinellosis. The antigen preparations were crude extracts of Trichinella spiralis. The Western blot profile characteristic of trichinellosis was characterized by comparing 60 sera from patients infected by Trichinella to 11 sera from healthy subjects, 51 sera from patients with other proven parasitic diseases (cysticercosis, schistosomiasis, strongyloidosis, fascioliasis, toxocariasis, liver amebiasis, anisakiasis, filariasis, toxoplasmosis, hydatidosis, or malaria), and 23 sera from patients with autoantibodies. Specific 43- to 44-kDa and 64-kDa bands were obtained with all of the sera from 51 patients with acute trichinellosis, in 4 out of 9 patients at the early stages of the disease, and in only 1 control patient, who had suspected anisakiasis and in whom trichinellosis could not be ruled out by muscle biopsy.     

Molecular cloning and expression of Cu/Zn-containing superoxide dismutase from Fasciola hepatica

The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.