Hepatica Excretory-secretory Antigens Research Articles

Increased specificity of Fasciola hepatica excretory-secretory antigens combining negative selection on hydroxyapatite and salt precipitation

A single and rapid method to obtain an antigenic fraction of excretory-secretory antigens (ESAs) from Fasciola hepatica suitable for serodiagnosis of fascioliasis is reported. The procedure consists in the negative selection of F. hepatica ESAs by hydroxyapatite (HA) chromatography (HAC; fraction HAC-NR) followed by antigen precipitation with 50% ammonium sulphate (AS) and subsequent recovery by means of a Millex-GV or equivalent filter (Fi-SOLE fraction). Tested in indirect ELISA, the Fi-SOLE antigens detected natural infections by F. hepatica with 100% sensitivity and 98.9% specificity in sheep, and 97.7% sensitivity and 97.7% specificity in cattle, as determined by ROC analysis. The SDS-PAGE and proteomic nano-UHPLC-Tims-QTOF MS/MS analysis of fractions showed that the relative abundance of L-cathepsins and fragments thereof was 57% in fraction HAC-NR and 93.8% in fraction Fi-SOLE. The second most abundant proteins in fraction HAC-NR were fatty-acid binding proteins (11.9%). In contrast, free heme, and heme:MF6p/FhHDM-1 complexes remained strongly bond to the HA particles during HAC. Interestingly, phosphorylcholine (PC)-bearing antigens, which are a frequent source of cross-reactivity, were detected with an anti-PC mAb (BH8) in ESAs and fraction HAC-NR but were almost absent in fraction Fi-SOLE.

Production of Monoclonal Antibody Against Excretory-Secretory Antigen of Fasciola hepatica and Evaluation of Its Efficacy in the Diagnosis of Fascioliasis.

Parasitological methods are not helpful for the diagnosis of fascioliasis in acute and invasive periods of the disease. Detection of coproantigens seems to be a suitable alternative approach in the diagnosis of fascioliasis. The present study aimed to develop a reliable antigen detection system, using monoclonal antibodies raised against excretory-secretory (ES) antigen of Fasciola hepatica, for the diagnosis of fascioliasis. Fasciola adult worms were collected from the bile ducts of infected animals. Species of the fluke was determined by polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR). ES antigen of F. hepatica was prepared. For production of monoclonal antibodies, mice were immunized with ES antigens of F. hepatica. Spleen cells from the immunized mice were fused with NS-1 myeloma cells, using polyethylene glycol. Hybridoma cells secreting specific antibody were expanded and cloned by limiting dilution. Moreover, polyclonal antibody was produced against F. hepatica ES antigen in rabbits. A capture enzyme-linked immunosorbent assay (ELISA) system, using produced monoclonal antibody, was designed and stool samples of infected animals along with control samples were tested by the system. The capture ELISA detected the coproantigen in 27 of 30 (90%) parasitologically confirmed fascioliasis cases, while 4 of 39 (10.25%) samples infected with other parasitic infections showed a positive reaction in this system. No positive reactivity was found with healthy control samples. Accordingly, sensitivity of 90% and specificity of 94.2% were obtained for the capture ELISA system. The results were compared with those obtained with commercial BIO-X ELISA, and a very good (kappa = 0.9) agreement was found between the commercial kit and the developed capture ELISA. Findings of this study showed that the produced monoclonal antibody has appropriate performance for the detection of Fasciola coproantigen in stool samples and can be appropriately used for the diagnosis of fascioliasis.

Detection of Fasciola hepatica and Fasciola gigantica common and uncommon antigens, using rabbit hyper immune serum raised against their excretory-secretory and somatic antigens.

Fasciolosis is an important neglected helminth disease caused by two liver flukes, Fasciola hepatica and Fasciola gigantica. The two species of Fasciola are usually different in their morphological and molecular features. They have also common and uncommon antigens in both their somatic and excretory secretory metabolites. In this study, we compared somatic and excretory-secretory (ES) antigens of F. hepatica and F. gigantica, by using rabbit hyper immune serum raised against these antigens. Adult worms were collected from bile ducts of infected animals and species of the fluke was confirmed by RFLP-PCR. ES and somatic antigens of both species were prepared. Rabbits were subcutaneously immunized with either ES or somatic antigens to produce antibodies against these antigens. SDS-PAGE pattern of F. hepatica and F. gigantica somatic antigens was similar and both of them revealed 30 protein bands, ranging from 18 to 180kDa. In contrast, SDS-PAGE pattern of ES antigen of the two species was different. While protein bands with molecular weight of 18, 27, 29, 48, and 62kDa were common in both species, bands of 19, 45, 55 and 58kDa were only noticed in F. hepatica ES antigen. Rabbit polyclonal antibodies, raised against F. hepatica and F. gigantica ES antigen, reacted with main five protein bands, 25, 27, 29, 62 and 67kDa and polyclonal antibodies raised against somatic antigens of both species reacted with three protein bands, 25, 27 and 72kDa. Thus, the 25, 27 and 29kDa protein bands may serve as immunodominant antigens, which might be considered for serodiagnosis of fasciolosis. Moreover, bands of 62 and 67kDa in ES antigen and 72kDa in somatic antigens of both species were immunodominant and might be suitable candidate for development of serological assays for diagnosis of fasciolosis.

An ultrasensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3).

A capture enzyme-linked immunosorbent assay (ELISA) using a new monoclonal antibody (mAb MM3) is reported for the detection of Fasciola hepatica excretory-secretory antigens (ESAs) in feces of infected hosts. The mAb MM3 was produced by immunization of mice with a 7- to 40-kDa purified and O-deglycosylated fraction of F. hepatica ESAs, which has previously been shown to be specific for the parasite. The specificity and sensitivity of the MM3 capture ELISA were assessed using feces from sheep and cattle. Sheep feces were obtained from a fluke-free herd (with most animals harboring other nematodes and cestodes), from lambs experimentally infected with 5-40 F. hepatica metacercariae and in some cases treated with triclabendazole at 14 wk postinfection (PI), and from uninfected control lambs. Cattle feces were collected at the slaughterhouse from adult cows naturally infected with known numbers of flukes (from 1 to 154) or free of F. hepatica infection (though in most cases harboring other helminths). The MM3 capture ELISA assay had detection limits of 0.3 (sheep) and 0.6 (cattle) ng of F. hepatica ESA per milliliter of fecal supernatant. The assay detected 100% of sheep with 1 fluke, 100% of cattle with 2 flukes, and 2 of 7 cattle with 1 fluke. The false-negative animals (5/7) were probably not detected because the F. hepatica individuals in these animals were immature (5-11 mm in length). As expected, coproantigen concentration correlated positively (r = 0.889; P < 0.001) with parasite burden and negatively (r = 0.712; P < 0.01) with the time after infection at which coproantigen was first detected. Nevertheless, even in animals with low fluke burdens (1-36 parasites), the first detection of F. hepatica-specific coproantigens by the MM3 capture ELISA preceded the first detection in egg count by 1-5 wk. In all sheep that were experimentally infected and then untreated, coproantigen remained detectable until at least 18 wk PI, whereas in sheep that were experimentally infected and then flukicide treated, coproantigen became undetectable from 1 to 3 wk after treatment. None of the fecal samples from sheep or cattle negative for fascioliasis but naturally infected with other parasites including Dicroelium dendriticum showed reactivity in the MM3 capture ELISA. These results indicate that this assay is a reliable and ultrasensitive method for detecting subnanogram amounts of F. hepatica antigens in feces from sheep and cattle, facilitating early diagnosis.

A novel Fasciola hepatica saposinlike recombinant protein with immunoprophylactic potential.

A member of the Fasciola hepatica saposinlike/NK-lysin protein family with lytic activity on human peripheral blood mononuclear cells and erythrocytes was recently described. The current study was designed to test the immunoprophylactic potential of this protein termed FhSAP-2 against infection with F. hepatica in rabbits. Two doses of 50 microg of recombinant FhSAP-2 (rFhSAP-2) emulsified in TiterMax were injected subcutaneously on the dorsal surface of 4 rabbits at 2-wk intervals. Four weeks after the second immunization, the rabbits were infected orally with 25 F. hepatica metacercariae. Four non-immunized-infected rabbits were used as controls. An enzyme-linked immunosorbent assay revealed high levels of antibodies to both rFhSAP-2 and F. hepatica excretory-secretory antigens by 2 wk after the first immunization, which were always significantly higher in immunized-infected rabbits than in control-infected rabbits. On the completion of the trial, vaccinated rabbits had 81.2% less flukes than controls. Moreover, F. hepatica egg counts in feces, as well as in bile collected from the gall bladders from vaccinated animals, were lower, 83.8 and 73%, respectively, compared with controls. The vaccinated rabbits also had significantly lower amounts of parasite antigen in stool and bile samples than controls. Last, evaluation of macroscopic liver lesions revealed that the rabbits vaccinated with rFhSAP-2 had milder lesions than the infected-control rabbits. These findings support the hypothesis that this novel rFhSAP-2 protein has immunoprophylactic potential against fascioliasis in rabbits including antifecundity and antipathology effects. This is the first report on experimental vaccination of rabbits against F. hepatica with a purified, defined, recombinant protein related to a member of the saposinlike protein family.

Optimized serodiagnosis of sheep fascioliasis by Fast-D protein liquid chromatography fractionation of Fasciola hepatica excretory-secretory antigens.

Current methods for the serodiagnosis of sheep fascioliasis show suboptimal sensitivity, specificity, or both. With the aim of developing an improved method, we fractionated native Fasciola hepatica excretory-secretory antigens (ESAs) by size-exclusion FPLC (fast protein liquid chromatography) on a Superdex 75 HR 10/30 column and then tested the serodiagnostic value of the antigens contained in each one of the 4 peaks obtained (peaks I-IV). Serodiagnostic value was assessed using sera from sheep naturally infected with F. hepatica (group A); sera from the individuals of a fluke-free herd (most of which also had other intestinal nematodes, lung nematodes, Moniezia spp., and/or Cysticercus tenuicollis) sera from a fluke-free herd (group B); sera from lambs experimentally infected with 10-40 F. hepatica metacercariae (group C); and sera from uninfected control lambs (group D). Enzyme-linked immunosorbent assay (ELISA) with peak I or II as target antigens (and to a lesser extent with peak III as target) showed reactivity with negative sera, so that it was not possible to establish cutoff values discriminating infected and uninfected animals. In contrast, when peak IV was used as target, a low cutoff value of 0.235 optical density units (mean + 4 SD) discriminated infected and uninfected animals, with 100% sensitivity and 100% specificity. ELISA with peak IV as a target identified infected animals (even animals that had received only 10 metacercariae) within 3-5 wk of infection and subsequently throughout the rest of the 14-wk monitoring period. In Western blotting analysis, again only the antigens contained in peak IV (range 7-40 kDa, under reducing conditions) were specific for diagnosis of infected animals. These results indicate that molecular sieving of F. hepatica ESAs by this procedure is a fast, simple, reproducible way of obtaining antigens useful for serodiagnosis of sheep fascioliasis.

Molecular cloning of a member of the Fasciola hepatica saposin-like protein family.

A 436-bp complementary DNA (cDNA) was isolated from an adult Fasciola hepatica cDNA expression library by screening with the serum from a rabbit infected with F. hepatica for 4 wk. The deduced amino acid sequence encoded by this cDNA is an 11.5-kDa polypeptide that has significant homology to F. hepatica NK-lysin protein, to several members of saposin-like or NK-lysin protein families, as well as 3 amoebapore precursors of Entamoeba histolytica. The most striking feature observed within this protein, denoted FhSAP-2, is the presence of 6 conserved cysteine residues arranged within 5 amphipathic alpha-helical domains and the presence of 7 hydrophobic residues in strictly conserved positions. Using enzyme-linked immunosorbent assay it was found that rFhSAP-2 is highly reactive with sera from rabbits infected with F. hepatica for 2-14 wk as well as with sera from humans with chronic fascioliasis. An anti-rFhSAP-2 rabbit antiserum reacted with F. hepatica excretory-secretory antigens by Western blot, revealing a major 11.5-kDa and 2 minor 46- and 67-kDa antigenic polypeptides. This suggests that FhSAP-2 may be an antigen released from cytoplasmic storage granules present within F. hepatica parasites. rFhSAP-2 also exhibits a strong lytic activity on human erythrocytes and peripheral blood mononuclear cells. This suggests that cell lysis could be 1 of the biological functions of this protein.

Fasciola hepatica-Induced Immune Suppression of Spleen Mononuclear Cell Proliferation: Role of Nitric Oxide

The aim of the present study was to evaluate the proliferative response of spleen mononuclear cells (Spm) to mitogens in rats infected withFasciola hepaticaand its correlation with Spm and peritoneal cell (PC) nitric oxide (NO) production on Days 1, 3, 7, 14, 30, and 60 postinfection. In addition, histological changes in the liver were also studied. The proliferative response to Con A ofF. hepatica-infected Spm was significantly decreased on Day 7 postinfection (P< 0.01). However, a pronounced increase of the proliferative response was detected from Day 3 until Day 60 when Spm were stimulated with LPS. In order to determine whether NO levels were modified duringF. hepaticainfections, we quantified nitrite in Spm and PC supernatants in cultures. Our results indicate a profound decrease of nitrite production by LPS-stimulated PC on the first and second weeks postinfection, and an increase in the levels of this mediator on LPS-stimulated Spm at the same postinfection time. TheF. hepaticaexcretory-secretory antigen (ESA) was in part involved in the decrease of nitrite production by LPS-stimulated PC. A mechanism to avoid an immune response during the first stages of liver penetration could explain the transient suppression observed in Spm proliferative responses. On the other hand, the decrease in NO production by rat-infected PC could also be one of the strategies of the parasite to avoid the potential killing effect of NO during peritoneal migration.

Inhibition of spleen cell proliferative response to mitogens by excretory-secretory antigens of Fasciola hepatica

The effect of Fasciola hepatica excretory-secretory antigen (ESA) on the proliferative response of spleen mononuclear (SpM) cells of normal rats to stimulation with mitogens has been examined. When ESA was added to normal SpM cells, there was a decrease in the proliferative response to concanavalin A (Con A) or lipopolysaccharide (LPS) in a dose-dependent manner. The addition of indomethacin, which blocks prostaglandin synthesis, or Nω-nitro- l-arginine methyl ester ( l-NAME) a specific inhibitor of nitric oxide (NO) synthase, had no effect on the ability of ESA to suppress the proliferative response to Con A. However, supplementation of the culture media with catalase, which degrades hydrogen peroxide (H 2O 2) or superoxide dismutase (SOD) to remove superoxide anion (O 2 −), resulted in a restoration of proliferation to Con A. When LPS was used as mitogenic stimulus no inhibitor added to the culture restored the proliferation. These results suggest that H 2O 2 and O 2 − are involved in the suppressor phenomenon induced by ESA in the T-cell proliferative events.

Modification of accessory activity of peritoneal cells from Fasciola hepatica infected rats.

The effect of Fasciola hepatica excretory-secretory antigen (ESA) was studied in the modulation of the accessory functions of peritoneal cells (PC) of rats infected with the parasite. PC rats infected with F. hepatica 7 and 14 days previously showed a marked decrease in phagocytic activity against Candida tropicalis (P < 0.007 and P < 0.004, respectively). The same effect was observed when the assay was carried out with PC from animals injected 7 days before with ESA (P < 0.001) including PC previously treated in vitro with ESA. To investigate the effect of ESA on the antigen presenting ability, PC of animals infected before transfer to syngeneic normal rats were stimulated in vitro with either F. hepatica whole antigen (FhWA), ESA or human serum albumin (HSA). Delayed type hypersensitivity (DTH) response to the different antigens studied over a 35 day period was negative in rats transferred with sensitised PC. When these animals were immunised with the corresponding antigen the DTH response became positive. A similar result was obtained in PC receptors from ESA inoculated rats and in vitro stimulated with FhWA or HSA. These data suggest that the alterations observed in the functions of peritoneal cells may, in part be due to the effect of the F. hepatica ESA.

Detection of antibodies to Fasciola hepatica excretory-secretory antigens in experimentally infected goats by enzyme immunosorbent assay

An ELISA with excretory-secretory (ES) antigens has been evaluated as a technique for the early detection of specific antibodies in Fasciola hepatica infections in goats. Goats were experimentally infected with 100 or 200 metacercariae of bovine origin and serum samples were taken periodically over 365 days. The ELISA test was performed with ES antigens (10 μg ml −1), a single dilution of sera (1:800) and anti-goat IgG conjugate (1:1000). ES specific antigens were detected in all infected goats between 15 and 30 days postinfection (PI) and maximum antibody levels were reached at 90 days PI. Positive antibody levels (significantly different from those of controls) were still found at 365 days PI. No significant differences were observed between goats infected with 100 or 200 metacercariae. In all infected goats, eggs appeared in faeces between 60 and 90 days PI. ELISA with ES antigens could be a feasible method for the early diagnosis of goat fasciolosis.

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.

Eosinophil differentiation in response to Fasciola hepatica and its excretory/secretory antigens

Bone marrow cells from mice infected with Fasciola hepatica, from mice injected with F. hepatica excretory/secretory (ES) antigens, and from uninfected or uninjected control animals were cultured in the presence of F. hepatica ES antigens or the eosinophil differentiation cytokine IL-5. Eosinophil maturation in cultures was assessed quantitatively by measuring eosinophil peroxidase (EPO) activity and qualitatively by visual appraisal in stained preparations over a week. It was found that the presence in all cultures (including those from control animals) of either ES antigens at an optimal concentration of 100 μml −1 (established in preliminary trials) or IL-5 at 500 units ml −1 led to enhanced EPO activity. EPO activity in cultures without IL-5 or ES antigens remained static or fell over the culture period. At day 3 in all cultures containing IL-5 or ES antigens, there was maintenance of, or only a slight decline in, the number of eosinophils that were present when cultures were initiated, and more of them were mature than at day 0 as evidenced by their EPO activity. However, there was a marked fall in eosinophil numbers in all cultures in the absence of IL-5 or ES antigens. The results indicate that F. hepatica ES antigens, like IL-5, stimulate eosinophil maturation in bone marrow with a consequent rise in EPO activity in the cells. Whether the antigen(s) acts directly or indirectly on the eosinophils or their precursors has yet to be established. Nevertheless, it seems clear that F. hepatica produces a molecule with a functionally similar effect to that of IL-5.

Identification of a 17-kilodalton Fasciola hepatica immunodiagnostic antigen by the enzyme-linked immunoelectrotransfer blot technique.

Sera obtained from human patients, calves, sheep, and rabbits infected with Fasciola hepatica were tested by the Falcon assay screening test enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) techniques with Fasciola hepatica excretory-secretory antigens in order to evaluate their immunodiagnostic potential. The study included sera from 13 patients infected with F. hepatica or a history suggesting fascioliasis, 5 patients infected and treated with bithionol or praziquantel (3 were cured with bithionol), 10 patients infected with Schistosoma mansoni, 6 infected with Trichinella spiralis, and 13 controls and sera from calves, sheep, and rabbits with a primary F. hepatica infection. By FAST-ELISA with F. hepatica excretory-secretory antigens, the serum samples from fascioliasis patients gave the highest absorbance values, and the schistosomiasis patient sera gave intermediate values compared with a normal human serum control. Also by FAST-ELISA, the values for serum from patients with fascioliasis decreased steadily after cure, reaching normal levels 20 to 47 weeks postcure. In contrast, the serum from two patients who had been treated but were not yet cured had high levels of antibodies for up to 3 years of infection. By EITB, the serum samples from humans, rabbits, cattle, and sheep with fascioliasis recognized two antigenic polypeptides of 17 and 63 kilodaltons (kDa) in the form of sharp bands. For humans, this recognition lasted for at least 3 years of infection. Sera from individuals with schistosomiasis mansoni or trichinosis or from normal controls did not recognize the 17-kDa F. hepatica antigenic polypeptide. However, serum from one human with S. mansoni and one with T. spiralis infection has slight bands in the 63-kDa region, suggesting cross-reactivity. Reactivity to the 17-kDa polypeptide was absent in fascioliasis patients at 1 year postcure. Reactivity to the 63-kDa polypeptide was significantly diminished in fascioliasis patients at 1 year postcure. The sera from rabbits with a primary F. hepatica infection also recognized both the 17- and 63-kDa antigenic polypeptides by week 4 of infection. Reactivity to both antigens diminished significantly 6 weeks postcure and disappeared by 8 weeks postcure. The sera from infected cattle and sheep recognized these two antigenic polypeptides by week 8 of infection. These studies suggest that the 17-kDa F. hepatica excretory secretory antigen is an excellent candidate for the immunodiagnosis of acute and chronic fascioliasis. Purification of this antigen and its application to quantitative serologic tests will permit further analysis of its predictive value to evaluate cure.