Schistosome antigen gp50 is responsible for serological cross-reactivity with Trichinella spiralis.

The 50-kDa component (gp50) present in Schistosoma mansoni eggs and secretions of the various life stages of the parasite was recognized by experimentally infected mice and by humans with S. mansoni, Schistosoma haematobium, and Schistosoma japonicum infection. All sera reacting with crude S. mansoni-soluble egg antigens (SEA) also reacted strongly with gp50 in enzyme-linked immunosorbent assay. No reactivity against gp50 was seen with sera from individuals without schistosomiasis, with the exception of sera from patients with Trichinella spiralis infection. All of 10 sera from patients with trichinellosis also reacted with schistosomes by immunofluorescence essentially recognizing testes, ovaries, ootype epithelium and ducts of the reproductive system. Cross-reacting antigens were seen in T. spiralis hypodermis, stichocytes and possibly germinal primordia using anti-gp50 monoclonal antibodies and anti-gp50-positive schistosomiasis patient sera. The results suggest that the anti-gp50 antibody response constitutes a significant part of the anti-SEA antibody response in infected individuals and is a major reason for the previously recognized serological cross-reactivity between T. spiralis and schistosome species.     

Schistosoma mansoni, S. haematobium, and S. japonicum: identification of genus- and species-specific antigenic egg glycoproteins

Immunoreactive egg glycoproteins of Schistosoma mansoni, S. haematobium, and S. japonicum which are genus- and species-specific, or react with sera of patients infected with other parasites, have been identified. Egg proteins were labeled with Iodine-125, and the concanavalin A-binding glycoproteins were immunoprecipitated with sera of patients infected with one of four species of Schistosoma or Trichinella spiralis, Taenia solium, Echinococcus granulosus, Entamoeba histolytica, or Wuchereria bancrofti. These immunoprecipitates were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Despite the strikingly different patterns of glycoproteins of the African species, the antibody immune responses of patients infected with S. mansoni and S. haematobium were found to be so similar that differentiation could not be established. In contrast, sera of patients infected with S. japonicum, S. mekongi, or parasites not of the genus Schistosoma, immunoprecipitated fewer of the major S. mansoni or S. haematobium glycoproteins. Likewise, antibody immune responses of patients infected with the Oriental schistosomes (S. japonicum and S. mekongi) could not be differentiated. Only a few quantitative differences were noted between our S. mansoni egg glycoprotein extract and a standardized soluble egg antigen extract. This study provides an explanation for the extensive cross-reactivity observed in diagnostic assays which utilize various fractions of schistosomal egg extracts as the antigen.     

Eosinophi-mediated destruction of Schistosoma mansoni eggs in vitro. II. The role of cytophilic antibody

Peritoneal exudative eosinophils obtained from Schistosoma mansoni-infected CBA/J mice cause morphological damage to isolated S. mansoni eggs in a 24 hr co-cultivation system in vitro. This egg-destructive activity was complement-independent and was abolished by trypsinization of the cells prior to co-cultivation. Trypsinized cells could be passively sensitized to renewed egg-destructive capacity by preincubation or co-gcultivation with immune sera, containing antibodies against a soluble egg antigenic preparation (SEA). Solid phase absorption of immune sera with SEA coupled to Sepharose 4B lowered the anti-egg antibody titers of these sera and eliminated their ability to sensitize trypsinized eosinophils. Sera from uninfected mice or from mice infected with Trichinella spiralis did not sensitize trypsinized cells. Addition of immune sera to eosinophil-rich cell populations obtained from uninfected mice also enhanced the egg-destructive capacity of these otherwise non-reactive cells. Therefore, eosinophil-mediated destruction of S. mansoni eggs may be directed by cytophilic antigen-specific factors in sera from S. mansoni infected hosts.     

Anti-idiotypic T lymphocyte responsiveness in murine Schistosomiasis mansoni

Pooled sera from CBA/J mice infected for greater than or equal to 16 weeks with the blood fluke Schistosoma mansoni were immunoaffinity purified using soluble schistosome egg antigens (SEA) coupled to Sepharose 4B. The bound and then eluted fraction was shown to contain only immunoglobulins and to have anti-SEA activity. These anti-SEA antibodies stimulated proliferation of lymph node cells from mice infected with S. mansoni for 8, 12, or greater than or equal to 16 weeks but not from uninfected mice. The cells stimulated by anti-SEA antibodies were nylon wool adherent, Thy-1.2+, L3T4+, Lyt-2-lymphocytes. Immunoglobulins without anti-SEA activity isolated from the sera of syngeneic uninfected mice were not stimulatory for cells from normal or infected animals. Thus the responding T cells appear to be stimulated by the idiotypes expressed on the syngenic anti-SEA antibodies. These data present evidence for anti-idiotypic cellular reactions in murine schistosomiasis that could play important immunoregulatory roles in this disease.     

Schistosoma mansoni: immunodiagnosis is improved by sodium metaperiodate which reduces cross-reactivity due to glycosylated epitopes of soluble egg antigen

ELISA with soluble egg antigen (SEA) from Schistosoma mansoni is widely used in the diagnosis of schistosomiasis, but cross-reactivity with other intestinal helminths, overestimating the true prevalence, represents a great limitation. The role of glycoproteins of SEA in cross-reactions was investigated. SEA was oxidized with sodium metaperiodate (SMP) in ELISA and immunoblot. One hundred schistosomiasis-negative individuals sera were submitted to SMP-ELISA improving the specificity from 73% without SMP treatment to 97% with SMP. On the other hand, 94 S. mansoni-positive sera were evaluated showing that 99% were positive in ELISA either with or without SMP treatment, indicating the maintenance of high sensitivity under SMP treatment. By immunoblot, 24 sera from persons with schistosomiasis and 10 sera from schistosomiasis-free persons were assayed under reducing and nonreducing conditions with or without SMP, looking for specific infection markers and cross-reactivity markers. Reactivity from positive sera showed that specific molecules were mainly low-molecular-mass antigens and seem to have predominant proteic epitopes. The unspecific molecules reacting with some schistosomiasis-negative individuals harboring other intestinal parasites (false-positive sera) were mostly larger than 60 kDa and seemed to be basically glycosylated. Glycosylated epitopes have an important role in cross-reaction and SMP can successfully be used to reduce the false reactivity of SEA with no decrease in sensitivity, especially in ELISA as an immunodiagnostic screening surveillance method, which is useful in areas of low schistosomiasis transmission.     

Lower faecal egg excretion in chemically-induced diabetic mice infected with Schistosoma mansoni due to impaired egg maturation

The effect of streptozotocin-induced diabetes mellitus was studied in mice infected with Schistosoma mansoni. Faecal egg excretion was lower in diabetic mice but worm load and total amount of eggs in the intestine tissue were equal to the control group. Evaluation of an oogram showed a great number of immature dead eggs and a low number of mature eggs in diabetic mice. It was therefore concluded that faecal egg excretion was lower in diabetic mice due to impaired egg maturation.     

Detection of IgG binding to Schistosoma mansoni recombinant protein RP26 is a sensitive and specific method for acute schistosomiasis diagnosis

We recently described the first recombinant Schistosoma mansoni protein RP26, which was capable of acute infection diagnosis. The aim of the present work was to further characterize the RP26 diagnostic properties in immunoblot and enzyme-linked immunosorbent (ELISA) assays. Testing sera from uninfected donors and sera from patients with acute or chronic Schistosoma infection by Western blot immunoassay revealed 100% specificity and 100% sensitivity for acute infection identification. Sera from uninfected, acute, and chronic schistosomiasis were also probed for IgG, IgG4, IgA, and IgM reactivity to RP26 plus soluble egg antigens (SEA) in ELISA. The mean IgG reactivity to RP26 by sera from acute schistosomiasis patients was significantly higher than the chronic ones. The IgG4, IgA, and IgM reactivities to RP26 were low and similar in both infected groups. The mean IgA and IgM reactivities to SEA were significantly higher in the group of acute compared to chronic group, whereas mean IgG4 reactivity was higher in chronic group. To estimate the specificity of Schistosoma infection diagnosis sera from patients infected with other different parasites were tested to detect IgG reactivity to RP26 and IgA and IgM reactivity to SEA. For IgA against SEA detection, 72% of sera were positive and 48% of sera were positive for IgM detection. Based on these results we can suggest that detection of sera IgG binding to RP26 is a sensitive and specific method for acute schistosomiasis diagnosis. Therefore, RP26 is a candidate for immunodiagnostic kit development.     

Demonstration of splenic auto-anti-idiotypic plaque-forming cells in mice infected with Schistosoma mansoni

Mice exposed to 35 cercariae of the human helminth Schistosoma mansoni develop chronic (greater than 16wk) infections characterized by immunoregulation of their cell-mediated granulomatous responses to schistosome eggs. Evidence was sought regarding the possible development of anti-idiotypic responses against the responses to soluble egg antigens (SEA). Sera were collected from CBA/J mice with chronic S. mansoni infections. Multiclonal idiotypic, anti-SEA antibody (id) was prepared from these pooled sera by affinity chromatography on an SEA immunoadsorbent column. Analysis of the id preparations by polyacrylamide gel electrophoresis demonstrated that this material contained only immunoglobulin heavy and light chains. A modified reverse plaque-forming cell (PFC) assay was developed to quantify anti-idiotypic (anti-id) PFC in spleen cell preparations from infected and age-matched control CBA/J mice. Expression of anti-id PFC began 2 to 3 wk after onset of egg production and continued throughout the course of infection. Positive selection of anti-id-reactive spleen cells by panning cell preparations from chronic mice on id-coated plates resulted in an enrichment of anti-id PFC in the id-adherent population. Conversely, the number of PFC reactive with SEA (id-producing PFC) was lowered by panning on id-coated plates. These data demonstrate the occurrence of anti-id responses during schistosomiasis mansoni. It is possible that such an immunoregulatory mechanism could play an important role in how an animal modulates the granulomatous response that leads to the formation of pathologic lesions and in the maintenance of this chronic infection.     

Schistosoma mansoni: detection and characterization of antigens and antigenemia by inhibition enzyme-linked immunosorbent assay (IELISA)

An inhibition enzyme-linked immunosorbent assay (IELISA) was used to detect the presence of schistosome antigens obtained from cercariae, adult worms, and eggs of the parasite. Using appropriate titers of Schistosoma mansoni infected mouse serum (IMS), it was possible to detect less than 10 ng/ml of schistosome antigen when added to phosphate-buffered saline (PBS, pH 7.2) or normal human serum (NHS). The sensitivity of the test was highly contingent on the number of experimental variables including antibody titer and antigenic source. The results of specificity studies were complicated. Although there was no cross-reactivity detected with other unrelated antigen preparations, extensive cross-reactivity between various schistosome species and "stage-specific" antigens was observed. The IELISA, utilizing IMS, can quantitate the degree of antigenic cross-reactivity, i.e., genus-specific and cross-reacting antigenic determinants. Soluble egg antigen (SEA) preparations obtained from S. mansoni and S. japonicum actually "cross-reacted" more than cercarial- and egg-derived antigens obtained from the same species (S. mansoni). This test also showed a 32-fold increase in specificity for the quantitative detection of specific antigenic determinants when monoclonal antibodies were used to restrict the heterogeneity of the measured response. The technique proved satisfactory for the quantification of parasitic burden in mice and the detection of active infections in humans. Circulating antigen disappeared with a t 1/2 of 72-96 hr after successful treatment.     

Expression of cross-reactive, shared idiotypes on anti-SEA antibodies from humans and mice with schistosomiasis

Immunoaffinity-purified antibodies against Schistosoma mansoni soluble egg Ag (SEA) from infected patients' sera differ idiotypically according to the donor's clinical form of the disease. The Id differ both by their ability to stimulate proliferation of anti-Id T cells and their recognition by anti-Id-specific sera. Also, mice infected with S. mansoni develop anti-Id T and B cell responses against mouse anti-SEA antibodies. We now show that anti-SEA antibodies from serum pools of chronic, but asymptomatic patients (AM1 and AM5) stimulate proliferation of spleen cells from mice infected with S. mansoni. However, AM8, anti-SEA antibodies from hepatosplenic patients, did not stimulate these spleen cells. The murine responses directly parallel patient studies where AM1 and AM5 Id-stimulated human PBMC, but AM8 Id did not. In competitive ELISA, using AM1 or AM-5-specific rabbit antisera or human anti-SEA mAb E5-specific rabbit antiserum, sera from mice infected for 8 and 16 wk (but not from uninfected mice) compete with AM1, AM5, or E5. These sera do not compete in the AM8/anti-AM8 competitive ELISA. Sera from 8-wk-infected mice inhibit more against AM1, AM5, and E5 than do sera from later infections, and anti-SEA immunoaffinity-purified antibodies from 8-wk-infected mice stimulate spleen cells from infected mice more than anti-SEA antibodies from sera of mice late in infection. However, spleen cells from more chronically infected mice are more responsive to either the murine or human anti-SEA antibody preparations than cells from mice with earlier infections. Both the ELISA data and lymphocyte responses indicate that anti-SEA antibodies from mice infected with S. mansoni for 8 wk bear Id cross-reactive with those expressed on anti-SEA antibodies from humans with chronic, asymptomatic schistosomiasis, but not those from hepatosplenic patients.     

An electrophoretic analysis of Schistosoma mansoni soluble egg antigen preparation

Schistosoma mansoni soluble egg antigen (SEA) has been examined electrophoretically. Sodium dodecyl sulfate (SDS) electrophoresis of SEA reveals an extremely heterogeneous protein composition. At least 18-20 distinct bands stain with Coomassie blue and at least 6 bands stain with periodic acid Schiff (PAS). Four of the PAS-positive bands stain only faintly with Coomassie blue. The estimated molecular weight range for these proteins is between 16,000 and 200,000 daltons. An acid soluble fraction was isolated from SEA which contained 5 of the 6 glycoproteins. An immunoelectrophoretic analysis of SEA reveals at least 5 distinct precipitin arcs when developed with serum from mice infected with S. mansoni for 16 weeks.     

Adult worm tegumental damage and egg-granulomas in praziquantel-resistant and -susceptible Schistosoma mansoni treated in vivo

The effect of treatment with praziquantel (PZQ) on the tegument of adult Schistosoma mansoni worms and on liver egg-granulomas has been examined in mice infected with PZQ-resistant and -susceptible parasite isolates. Two PZQ-resistant S. mansoni isolates, one selected by passage in the laboratory under drug pressure and one from Senegal established from eggs excreted by an uncured patient, were compared with PZQ-susceptible control isolates. Scanning electron microscopic observations on the tegument of Schistosoma adult worms treated in vivo with PZQ showed that more severe damage was inflicted by PZQ on susceptible worms than on drug-resistant worms. Observations on the pathology of Schistosoma egg-granulomas in the liver of infected mice after treatment with PZQ indicated that eggs from susceptible control isolates were more sensitive to PZQ than those from drug-resistant isolates.     

Characterization of a purified glycoprotein from Schistosoma mansoni eggs: specificity, stability, and the involvement of carbohydrate and peptide moieties in its serologic activity

A major egg glycoprotein (MEG) was purified from a crude soluble extract of Schistosoma mansoni ova (Egyptian strain) by successive steps of lectin affinity and ion-exchange chromatography. Radioiodinated MEG exhibited a single precipitation band upon immunodiffusion against antiserum from chronically infected mice, and ran as a single band on PAGE (Rf 0.38) and SDS-PAGE (Rf 0.36). Its estimated m.w. was 70,000. The degree of stage and species specificity of MEG and the effect of various treatments on its serologic reactivity were determined by radioimmunoassay (RIA). A low degree of cross-reactivity between MEG and similarly prepared soluble antigens from adult worms and cercariae was demonstrated by RIA inhibition tests, whereas a high degree of cross-reactivity was found between MEG and a crude soluble S. haematobium egg antigen. In similar RIA inhibition tests, the Puerto Rican S. mansoni had a lower degree of cross-reactivity with S. haematobium than the Egyptian strain. MEG was four times more abundant in SEA from a Puerto Rican strain of S. mansoni than in SEA from the Egyptian strain. The serologic reactivity of MEG was stable to heat at 100 degrees C for 60 min, to 0.1 N NaOH or HCl, and to 10% TCA. Treatment of MEG with pronase caused a limited fragmentation of the molecule and some loss of its serologic reactivity. Periodate oxidation resulted in a substantial loss of molecular mass and of serologic reactivity, leaving a low residual activity that is only partially cross-reactive with the bulk of MEG. These results suggest the importance of both carbohydrate and peptide moieties of MEG for its serologic reactivity.     

Early detection of Schistosoma mansoni infection by touchdown PCR in a mouse model

A detection assay for Schistosoma mansoni DNA in mouse serum samples based on touchdown PCR was developed and evaluated. The serum samples could be assayed directly without the need to extract DNA. No cross reactions between S. mansoni and related species inducing human schistosomiasis were observed. After the infection, mouse sera and feces were collected for 8 weeks. Anti-worm antigen IgG and anti-soluble egg antigen IgG were detected in the sera at 6 weeks post-infection by ELISA. The parasite's eggs were detected in the feces at 8 weeks. In contrast, S. mansoni DNA was detected in the sera at 2 weeks post-infection. These data suggest that touchdown PCR is a potential tool for the early diagnosis of S. mansoni infection.     

Schistosoma mansoni adult microsomal antigens, a serologic reagent. II. Specificity of antibody responses to the S. mansoni microsomal antigen (MAMA)

The purified Schistosoma mansoni adult microsomal antigen, MAMA, was used in the quantitative single-tube kinetic dependent enzyme-linked immunosorbent assay (k-ELISA) to measure antibody levels of various human patient sera. The 511 serum specimens tested were from patients with both homologous and heterologous infections. Sera from U.S., Egyptian, Brazilian, and Puerto Rican patients infected with S. mansoni reacted strongly with MAMA. Chinese patients infected with S. japonicum, and Nigerians or Egyptians infected with S. haematobium produced much lower responses to this antigen than those infected with S. mansoni. Sera from patients with echinococcosis, filariasis, paragonimiasis, clonorchiasis, trichinosis, amebiasis, and hepatitis and from healthy uninfected control individuals generally contained no detectable antibodies against this antigen. The S. mansoni adult microsomal antigen, MAMA, therefore, appears to be a highly potent and specific reagent for the serodiagnosis of S. mansoni infections.     

Immune responses during human Schistosomiasis mansoni. XI. Immunologic status of patients with acute infections and after treatment

Sixteen patients, 8 to 30 yr of age, with acute (toxemic) phase schistosomiasis mansoni were studied immunologically within 2 to 3 mo of their exposure to Schistosoma mansoni cercariae, and were monitored after chemotherapy. Total leukocyte levels and peripheral blood eosinophilias were higher in these patients than in similar individuals with chronic schistosomiasis mansoni. In contrast to chronic patients, the eosinophilias of the acute cases were decreased rather than elevated upon treatment. Total lymphocyte population (T and B cell) percentages were not altered during acute infection. Lymphoid subset (T3+, T4+, and T8+) analysis revealed elevated levels of both T4+ and T8+ cells. In vitro blastogenic responses of peripheral blood mononuclear cells (PBMN) to heterogeneous schistosome-derived antigens (eggs, SEA; adult worms, AW; and cercariae, CERC) were evaluated. SEA responsiveness was considerably higher than that of patients with chronic S. mansoni infections. The ratios of SEA to AW responses in acute cases gave a mean of 2.0, as opposed to 0.5 for a comparable group of chronically infected patients. The sera of most acute patients already contained suppressive factors that specifically decreased schistosomal antigen-induced PBMN blastogenesis. Chemotherapy of acute cases lead to a diminution of PBMN responsiveness to SEA and CERC. Treatment of patients with chronic infections lead to the elevation of such responses. PBMN from patients with acute infections produced lymphokine leukocyte inhibition factor upon exposure of the cells to SEA but not AW. A similar pattern was true for production of the lymphokine activity mitogenic factor. Levels of antibody in sera of acutely infected patients against SEA, CERC, and AW were considerably higher than levels in sera of chronically infected patients matched for age and intensity of their infections. These high antibody titers persisted for at least 6 mo after treatment, and were unrelated to the intensity of infection. The immunologic status of these patients with acute schistosomiasis mansoni differed considerably from patients with chronic infections. These findings re-emphasize the immunoregulatory events that apparently develop upon continued exposure to schistosomes and their products during chronic infection.     

The use of lyophilized Schistosoma mansoni eggs as antigenic particles in a radioimmunoassay

Lyophilized eggs of Schistosoma mansoni, when incubated briefly with serum from infected mice, bind antibodies, as made evident by subsequent binding of fluorescein labelled anti-IgG or 125I-labelled Protein A. On the basis of these findings, a radioimmunoassay was devised which employs whole lyophilized eggs (500 or 250 eggs/serum sample) as antigenic particles and 125I-labelled Protein A as a probe for antibody binding. Only 10 microliters of serum are required to obtain 90% of the maximal binding. Kinetic studies indicated that 70% of the maximal seropositivity develops in mice between five and six weeks after a light infection, reaches a maximum at eight weeks and fluctuates around a high plateau thereafter. Pre-incubation of the test serum with soluble egg antigen (SEA) considerably inhibits antibody binding to the eggs, suggesting that SEA-like antigens participate in the reaction.     

A monoclonal antibody from infected mice to a Schistosoma mansoni egg proteinase

A number of monoclonal antibodies were obtained by fusion of SP2/0 myeloma cells and spleen lymphocytes from mice infected with Schistosoma mansoni. These antibodies were tested for their ability to inhibit acidic, thiol-dependent proteinases previously isolated from Schistosoma mansoni eggs and adult worms. One of the monoclonal antibodies isolated inhibits egg proteinase activity measured in vitro with the use of a low m.w. synthetic substrate. This antibody, which is an IgG1 isotype, does not appreciably inhibit an acidic, thiol-dependent proteinase obtained from the adult stage of Schistosoma mansoni. Immunocytochemical methods with the monoclonal antibody have been used to localize the egg proteinase within a set of "penetration" glands in the unhatched miracidium.     

Isolation of a polysaccharide antigen from Schistosoma mansoni eggs.

A polysaccharide antigen was isolated from Schistosoma mansoni egg homogenates by the phenol procedure. The crude preparation (CPEA) contained at least two antigens. The more purified antigen (PEA) was isolated by sequential enzymatic treatment of CPEA with DNase, RNase, Pronase, and alpha-amylase. PEA was resistant to boiling, freezing and thawing, mild acid and alkali, and chloroform, but was destroyed with periodate. It gave a positive reaction with anthrone reagent. PEA was eluted in the wash fraction from a DEAE cellulose collumn and in the void volume of a Sephacryl 200 column. After immunoelectrophoresis and polyacrylamide electrophoresis there was little or no migration. Amino acid analysis failed to reveal ninhydrin-positive material in the a hydrolyzate of PEA. These resluts suggested that PEA is a neutral polysaccharide with a m.w. of more than 200,000 and contains no amino acids or hexosamine. Antibodies against PEA were detected in sera obtained from mice infected with S. mansoni. PEA is different from previously described antigens derived from schistosome eggs.     

Mice infected with Schistosoma mansoni generate antibodies to LacdiNAc (GalNAc beta 1-->4GlcNAc) determinants.

Schistosoma mansoni is a parasitic trematode infecting humans and animals. We reported previously that adult S. mansoni synthesizes complex type biantennary N-glycans bearing the terminal sequence GalNAc beta 1-->4GlcNAc-R (lacdiNAc or LDN). We now report that mice infected with S. mansoni generate antibodies to LDN, as assessed by ELISA using a synthetic neoglycoconjugate containing LDN sequences. Sera of infected mice, but not uninfected mice, contained primarily IgM and low levels of IgG toward LDN. Interestingly, these antibodies also recognize bovine milk glycoproteins, which are known to express LDN sequences. The anti-LDN in sera of infected mice were affinity purified on immobilized bovine milk glycoproteins and shown to specifically bind LDN. An IgM monoclonal antibody (SMLDN1.1) was derived from the spleens of S. mansoni infected mice and shown to specifically bind LDN determinants. Immunoblots with affinity purified anti-LDN and SMLDN1.1 demonstrate that LDN sequences occur primarily on N-glycans of numerous glycoproteins of adult S. mansoni. LDN sequences are also expressed in many glycoproteins from S. japonicum and S. haematobium. The availability of antibody to LDN determinants should aid in defining the roles of these glycans in helminth and vertebrate biology.