Dot-Blot Immunoassay of Fasciola gigantica Infection using 27 kDa and Adult Worm Regurge Antigens in Egyptian Patients

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.

diagnostic efficacy of Fasciola gigantica coproantigen in naturally infected cattle and buffaloes

Monoclonal antibody sandwich ELISA was used detect F. gigantica coproantigen in 33 selected animals according to the faecal history to evaluate sensitivity and specificity by using three different rabbit hyper-immune sera; Copro HIS, Egg HIS and ES HIS. The results showed that Copro HIS and ES HIS detected F. gigantica coproantigen in faecal of naturally infected cattle and buffaloes, but egg HIS failed. The 26-28 KDa coproantigen proved sensitivity [81.8%] and specificity [90.9%] in diagnosis of fascioliasis. Also, there was a positive statistical significance between number of F. gigantica egg per gm faeces [EPG] and mean sandwich ELISA OD. values for copro antigen. For diagnostic value of F. gigantica coproantigen in comparison with ES antigen, EITB was done on field sera of cattle and buffaloes of known faecal history. The F. gigantica coproantigen bands of 27.6 and 72.1 KDa were specific for diagnosis animal fascioliasis, but the 72.1 KDa was less sensitive than the 27.6 KDs. The immunoblotting reaction was more intensive than with fractionated ES antigen than with fractionated coproantigen

Efficacy of some Fasciola gigantica antigens

The present study tested the antigenic relationship between the three Egyptian strain of Fasciola gigantica antigens; coproantigen, excretory-secretory and egg antigens, versus their related hyper-immune sera and select the most specific one. By using SDS-PAGE, a structural homology was demonstrated in F. gigantica ES and egg antigens. This homology was resided in the components of the similar molecular weights between both antigens. When no cross-reaction was recorded with the coproantigen, the intense cross-reaction occurred between ES and egg antigens in ELISA technique. This was attributed to the presence of common bands at 18.0, 20.4 and 27.6 KDa in between them. Consequently, the F. gigantica coproantigen and Copro HIS reflected the lowest level of the cross-reaction with the other evaluated F. gigantica antigens. The cross-reaction elucidated in the present study between the F. gigantica ES and egg antigens was mainly at the low serum dilutions. The distinction between the specific and the cross-reactive binding activities was clearly marked with the highly diluted sera

Prepatent detection of Fasciola gigantica infection in bovine calves using metacercarial antigen.

Metacercarial antigen of Fasciola gigantica was evaluated for early immunodiagnosis of experimental bovine fasciolosis using ELISA and Western blot. In ELISA, the experimental F. gigantica infection was detected as early as 2 weeks post-infection (WPI). The gradual increasing trend of antibody level was observed from 2 to 7 WPI, followed by a plateau, which was maintained up to 14 WPI. In Western blot, sera from experimentally infected calves recognized one distinct polypeptide of 21 kDa in fractionated metacercarial antigen as early as 10th day post infection. From 2 WPI, more polypeptide bands were reacting. Recognition of these protein bands persisted till the end of the experiment (14 WPI). Cattle sera collected from the field showed 34.5% seroprevalence of fasciolosis by ELISA using MAg. Comparative immunoblot studies of metacercarial antigen with anti-Gigantocotyle explanatum and anti-Paramphistomum epiclitum sera revealed that 21 and 25 kDa polypeptides of metacercarial antigen did not cross-react with any of these sera and appear to be unique to F. gigantica and having the desirable qualities of early and specific immunodiagnosis.

Cross reactivity between ES and somatic antigens of Fasciola spp in enzyme linked immunosorbent assay

Excretory-secretory [ES] and somatic antigens of Fasciola hepatica and Fasciola gigantica were prepared from freshly collected flukes. Laboratory bred rabbits were immunized with antigens for preparation of antisera. ES antigens of both species showed strong positive reaction with antisera raised against ES and somatic antigens of parasite. Somatic antigens of both species also showed strong positive reaction with antisera raised against somatic and ES antigens of parasite. In homologous combination of antigens and antisera higher enzyme linked immunosorbent assay [ELISA] values was observed in comparison with heterologous combination, so it was concluded that ES and somatic antigens of Fasciola spp have strong cross reaction with each other but the antigenic materials of ES and somatic products of parasite are not completely the same

Serum interferon-gamma and interleukins-6 and -8 during infection with Fasciola gigantica in cattle and buffaloes

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.

Partially purified fraction [PPF] antigen from adult fasciola gigantica for the serodiagnosis of human fasciolisis using dot - Elisa Technique

Human fascioliasis has been reported in many countries, including Iran. Various techniques have been evaluated for diagnosis of human fascioliasis using different antigens. We evaluated Fasciola gigantica partially purified fraction antigen [PPF] isolated from sheep's liver fluke for the diagnosis of human fascioliasis. Materials and Two hundred sixty-one sera were collected from 104 patients living in an area endemic for human fascioliasis, from 89 non-fascioliasis patients living in a non-endemic area, and from 68 healthy individuals. Micro-ELISA was used in the evaluation of the sensitivity and specificity of Dot-ELISA. With a 1:800 sera dilution as the cut-off titer, the sensitivity of the Dot-ELISA test in diagnosis of human fascioliasis was 94.23% and the specificity was 99.36%. Dot-ELISA using PPF antigen is a sensitive and specific method for diagnosis of human fascioliasis that is also rapid and inexpensive

Immunodiagnosis of human fascioliasis using an antigen of Fasciola gigantica adult worm with the molecular mass of 27 kDa by a dot-ELISA.

Immunodominant antigens of an approximate molecular mass of 27 kDa (FG 27) were obtained from an excretory-secretory product of adult Fasciola gigantica by a simple continuous-elution method. A dot-ELISA using the FG 27 antigen was developed for the detection of specific antibodies from patients infected with F. gigantica. Control sera were obtained from patients with other parasitic infections and healthy volunteers. The accuracy, sensitivity, specificity, and positive and negative predictive values were 98.2%, 100%, 97.4%, 76.9% and 100%, respectively. This dot-ELISA is a specific, sensitive and easy to perform method for the rapid diagnosis of fascioliasis, particularly when more complex laboratory tests are unavailable.

Localization of antigenic molecules of adult Fasciola gigantica using monoclonal antibodies against the parasite tegumental antigen.

In this study, monoclonal antibodies were developed from the partially purified surface tegumental antigens of F. gigantica. Nine MoAbs: 2G11, 1G2, 1B12, 2G2, 2G5, 3C6, 3G2, 3G3 and 3F6 were used for anatomical localization of adult F. gigantica. The reaction was demonstrated by Avidin-Biotin method. The results revealed that among the sections stained with non-immune sera and control group, there were no reaction products on either the tegument or the cecal epithelium. The only brownish areas were the vitelline glands. In the sections stained with immune sera, brownish reaction products appeared on the surface membrane, the spine membrane, the cecal lumen and its epithelial cells. The experiment sections of nine monoclonal antibodies revealed that the reaction occurred mainly on the tegument of the adult worm which covered its surface and spine.

Production and characterization of a monoclonal antibody against recombinant glutathione S-transferase (GST) of Fasciola gigantica.

A monoclonal antibody (MoAb) against a recombinant glutathione S-transferase (rGST) of F. gigantica was produced in BALB/c mice. Reactivity and specificity of this monoclonal antibody was assessed by ELISA and immunoblotting. Six stable clones, namely 3A3, 3B2, 3C6, 4A6, 4B1 and 4D6 were obtained, All these MoAb reacted with rGST and native GST at a molecular weight of 28 kDa and found to be IgG1, kappa-light chain isotypes. These MoAb cross-reacted with Schistosoma mansoni and Schistosoma japonicum antigens at molecular weights of 28 and 26 kDa, respectively, but no cross-reactions were detected with antigens of Eurytrema and Paramphistomum spp. The localization of GST in metacercaria, 7-week-old juvenile and adult F. gigantica was performed by immunofluorescence technique, using MoAb as well as polyclonal antibody (PoAb) to the native protein as probes. In general, all clones of MoAb gave similar results and the pattern was quite similar to staining by PoAb. The fluorescence was intense, which implied the presence of a high concentration of GST in the parenchymal tissue in all stages of the parasite. However, the parenchymal cells were not evenly stained which implied the existence of subpopulations of this cell type with regard to GST production and storage. In addition, in adult and juvenile stages a moderate fluorescence was present in the basal layer of the tegument, while light fluorescence was observed in the caecal epithelium, cells in the ovary, testis and vitelline gland of the adult. In the metacercaria stage, in addition to parenchymal tissue, the tegument and tegumental cells were stained relatively more intense with MoAb and PoAb than in other stages.

Production of monoclonal antibodies against partially purified surface tegument antigens of Fasciola gigantica.

Monoclonal antibodies (MAbs) were produced by the in vitro fusion of Balb/C mice spleen cells immunized with partially purified surface tegument antigens of Fasciola gigantica. The surface membrane and tegument antigens were purified by using gel filtration chromatography. SDS-PAGE performed on the processed proteins demonstrated that the proteins had molecular weights of 20 to 97 kDa. In this study, fifteen monoclonal antibody clones were selected from the hybridoma clones, namely: 1B7, 1B11, 1B12, 1C9, 1D4, 1G2, 1H7, 2B6, 2C3, 2C9, 2D11, 2F11, 2G2, 2G5, and 2G11. They were evaluated by immunoblot assay and were differentiated into two groups. In the first group were found 60 and 38 kDa proteins; in the second group were found 66, 60, and 38 kDa proteins. All were found to secrete IgM, kappa light-chain antibodies. These MAbs were tested for their cross-reactivity with other trematodes commonly found to infect cattle and man. All of these MAbs showed some degree of cross-reactivity with other trematode species.

Fascioliasis in Vietnam.

A confirmed diagnosis of human fascioliasis was rare in Vietnam until 1978 when two cases were reported in humans. Since 1997, we have confirmed 500 cases of human fascioliasis. The majority of cases come from the central provinces of Vietnam: Da Nang, Quang Ngai, Binh Dinh, Phu Yen and Khanh Hoa. Patients were treated in hospitals in Ho Chi Minh City. All had high peripheral blood eosinophilic counts (16-70%) and positive serology with Fasciola gigantica antigen with positive titers of 1/1,600 to 1/12,800. We are unsure whether this represents an endemic pattern of disease or whether improved specific laboratory tools now facilitate better diagnosis. It is also possible that with changes in environmental factors and in the number and breeds of herbivorous domestic animals, Fasciola is increasing in frequency and easily contaminates the food.

Identification of circulating antibodies in fasciolosis and localization of 66 kDa antigenic target using monoclonal antibodies.

We identified three specific circulating antibodies in serum of cattle naturally infected with Fasciola gigantica. Two of the antibodies were found to react specifically to 97 and 66 kDa antigenic molecules of adult worm tegumental membrane extract. The third antibody was identified by the reaction with 26-28 kDa molecule of the excretory/secretory antigens. Monoclonal antibody against 66 kDa protein was developed and used for localization of its antigenic target in adult worm frozen sections. The experiment demonstrated that 66 kDa protein is a component on the outer surface membrane and on the membrane lining of the caecal epithelial of adult worm. The 66 kDa antigen was considered as a promising candidate for immunodiagnosis and vaccine.

Detection of fasciola-specific excretory/secretory [e/s] protein fraction band [49.5 kda] and its utilization in diagnosis of early fascioliasis using different diagnostic techniques

The objective of the present work was to evaluate Fasciola E/S antigens for diagnosis of early fascioliasis utilizing different diagnostic techniques. Using enzyme-linked immunoelectrotransfer blot [EITB], Fasciola-specific E/S protein fraction band [49.5 kDa] was determined and electroeluted. The monospecific antibodies against this specific fraction band were prepared by immunizing pathogen-free rabbit. Assessment of the prepared monospecific antibodies in diagnosis of human fascioliasis was performed through the detection of E/S coproantigens by enzyme-linked immunosorbent assay [ELISA] in stool eluates obtained from patients with confirmed fascioliasis, other parasites as well as from other healthy individuals. Serum samples were collected and tested to detect serum antibodies against Fasciola E/S antigen using EITB and counter immunoelectrophoresis [CIEP]. Analysis of Fasciola adult worm E/S products by SDS/PAGE revealed a number of bands, the molecular weight [MW] of which ranged from 14-200 kDa with 3 major bands [27.5, 32.5 and 55 kDa]. Fasciola E/S 49.5 kDa protein fraction proved to be specific of F. gigantica. Cross reaction with S. mansoni was observed at higher MW [110-120 kDa]

Diagnosis of cattle fasciolosis by the detection of a circulating antigen using a monoclonal antibody.

A monoclonal antibody (MoAb) 1C12 that reacts with a 66 kDa surface tegumental (ST) antigen of adult worms of Fasciola gigantica was used to detect circulating antigen in sera of experimentally and naturally infected cattle. A combination of rabbit anti ST-antigens and MoAb 1C12 were used to capture and detect the circulating antigen in sandwich ELISA. The dilutions of 1:1,000 of rabbit anti ST-antigens and 1:100 for MoAb 1C12 were used to reduce cross-reactivity with other trematodes' antigens. The circulating antigen of F. gigantica was demonstrated in sera of all experimentally infected animals as early as the first week after the infection, and it remained detectable until the experiment was terminated at week 32 after the infection. Of the 97 serum samples from naturally infected cattle, the sensitivity of 86.6% was observed when the cut-off point was calculated from 32 serum specimens from uninfected control calves. The sensitivity increased to 100% when the commercial fetal calf and trematode-free baby calves sera were used for calculation of the control cut-off point. Based on these results, the combination of rabbit anti ST-antigens and MoAb 1C12 sandwich ELISA appeared to be sensitive, specific, and applicable in the immunodiagnosis of fasciolosis in cattle for epidemiological study and monitoring of chemotherapeutic efficacy.

Specific monoclonal antibodies to Fasciola gigantica.

Monoclonal antibodies (mAbs) were produced against soluble metabolic products (excretory/secretory; ES) of the liver fluke, Fasciola gigantica. The ES antigen was obtained from spent culture medium in which the adult flukes had been maintained in vitro. From two cell fusions, the mAbs produces were exclusively associated with either IgG or IgM isotypes. When screened against a panel of homologous and heterologous parasite antigens by indirect ELISA, two mAbs were highly specific for F. gigantica. The remainder cross-reacted extensively with other parasites. Results from immunoblotting and immunofluorescence exhibited two patterns of reactivity. The first group of mAbs which recognized the multiple bands between > 14.4-27.5 kDa gave extremely bright fluorescence over all major muscular systems, vitelline gland, testes and intestinal caeca. The second group which reacted with the 185 kDa band showed a bright fluorescence over a thinner muscular layer underlying the tegument, intestinal caeca and testes.

Development and characterization of monoclonal antibodies against excretory-secretory antigens of Fasciola gigantica.

Monoclonal antibodies (MAbs) directed against Fasciola gigantica excretory-secretory (ES) antigens were developed from BALB/c mice. Four were selected for further study, from the panel of hybridomas. The antigen specificities of these MAbs were characterized and localized by enzyme-linked immunoeletrotransfer blot (EITB) and immunoperoxidase technique. The target epitopes of these MAbs are 66 kDa protein (MAb 2D10), 66 and 27-26 kDa proteins (MAbs 5D10 and 4F5) and 27-26 kDa protein (MAb 2D9). MAb 2D9 reacted to the antigenic components of the luminal content and epithelial cell lining the cecum, whereas MAb 2D10 reacted specifically to the antigens of the tegument and surface membrane. It was found that all MAbs cross-reacted to various degrees with the antigens extracted from Schistosoma mansoni, S. mekongi, S. spindale and Paramphistomum spp. However, when MAbs were diluted to 1:100 or 1:400 significant reduction of their cross-reactivities was observed.

Detection of circulating antigens in blood to evaluate treatment of fascioliasis

This work was designed to assess cure of human fascioliasis after triclabendazole treatment by detection of Fasciola antigen by a modified double antibody sandwich ELISA technique. The results showed that the test detected the antigen in the sera of all studied cases before treatment, while no antigen was detected after treatment. The results suggested that antigen detection provides an accurate tool for diagnosis as well as the assessment of cure

Comparison of adult somatic and excretory-secretory antigens in enzyme-linked immunosorbent assay for serodiagnosis of human infection with Fasciola gigantica.

Adult somatic antigen extract of Fasciola gigantica was compared with excretory-secretory (ES) antigen in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of human fascioliasis gigantica. The absorbance values in ELISA using the adult somatic antigen were not significantly different from the values obtaining using ES antigen (p > 0.05). The diagnostic sensitivity, specificity and positive and negative predictive values of the test using adult somatic extract as antigen were 100%, 98%, 70% and 100%, respectively. On the other hand, these values of the test using adult ES antigen were 100%, 99.3%, 87.5% and 100%, respectively. It appears that both somatic and ES antigens are effective antigens for use in the serodiagnosis of human fascioliasis gigantica.

Immunodiagnostic moieties in somatic and excretory/secretory antigens of Fasciola gigantica.

Immunodiagnostic potential fractions in fluke antigens and excretory/secretory (E/S) antigen of F. gigantica have been identified using double immunodiffusion (DID), immunoelectrophoresis (IEP) and sodium dodecyl sulphate (SDS) PAGE electrophoresis. Hyperimmunised rabbits elicited considerable level of antibody response with few precipitin lines showing reaction of identity in DID suggesting presence of some common fractions in all antigens. Mature fluke somatic (MFSAg) and immature fluke somatic (IMFSAg) antigens showed exactly similar pattern of precipitin lines in both DID and IEP. Antibodies to experimentally infected rabbits with F. gigantica were also detected by DID using MFSAg and E/S antigens at 4 weeks post infection. However, out of 6-7 fractions obtained from Sephadex G-200 column chromatography in MFSAg and IMFSAg antigens only F3, F4 and F5 along with E/S antigen were able to detect antibodies by DID. Mature fluke and E/S antigen separated out in 9 bands in the range of 12 to 95 kDa and 5 bands in the range of 12 to 29 kDa respectively of which proteins of molecular weight 12, 15, 18, 28 and 29 kDa were common in both antigens. Therefore, it appears that immunodiagnostic fractions of antigens may be present in between 12 and 29 kDa.