Irradiated Cercariae Research Articles

Protective immunization of mice against Schistosoma mansoni with purified adult worm surface membranes

Immunity to Schistosoma mansoni in the mouse was induced by vaccination with adult worm surface membrane (mb-S). Of several adjuvants tested, including Freund's, BCG and alum, 50 micrograms of saponin per mouse given subcutaneously with the antigen was the easiest to administer, and gave consistent protection, approaching levels usually seen in our mouse model after exposure to irradiated cercariae. An antibody response to the schistosomular surface was detected in mice immunized with mb-S plus saponin which was predominantly anti-polypeptide, not anti-carbohydrate, and thus similar to the antibody response of mice exposed to irradiated cercariae. The level of antibodies to Mr 90,000 and 38,000 schistosomular surface antigens as well as to Mr 25,000 adult surface membrane antigen was significantly correlated with the presence of protection.

Variable species and stage specificity of schistosomulum surface epitopes recognized by mice vaccinated with highly irradiated cercariae

Antibodies from mice vaccinated with highly irradiated Schistosoma mansoni or S. haematobium cercariae were used to characterize schistosomulum surface epitopes which were found to be diverse in their species and stage specificities. The epitopes recognized on the Mr greater than 200,000 and 15,000 schistosomulum surface antigens of S. mansoni and the Mr greater than 200,000 schistosomulum surface antigen of S. haematobium were found to be cross-specific whereas those on the Mr 38,000, 32,000 and 20,000 schistosomulum surface antigens of S. mansoni and the Mr 35,000, 30,000 and 24,000 schistosomulum surface antigens of S. haematobium were only immunoprecipitated by homologous antibody and are thus possible targets of the protective species-specific immunity stimulated by highly irradiated cercariae. The epitopes recognized on the Mr greater than 200,000 and 38,000 antigens of S. mansoni were shown to cross-react with both the egg and the adult worm whereas those on the Mr 32,000 and 20,000 antigens only cross-reacted with the adult worm, and those on the Mr 15,000 antigen cross-reacted with neither the adult worm nor the egg. In addition the epitopes on the Mr 38,000 and 32,000 antigens were demonstrated to be polypeptide in nature. Those on the Mr greater than 200,000, 20,000 and 15,000 antigens, on the other hand, could not be conclusively defined.

The Ability of Fractionated Sera from Animals Vaccinated with Irradiated Cercariae of Schistosoma mansoni to Transfer Immunity to Mice

To study the role of IgG and IgM isotypes found in sera of mice or rabbits immunized with irradiated cercariae in schistosome immunity, the respective sera were fractionated by protein A chromatography. Both the protein A-bound and unbound fractions of vaccinated mouse serum (VMS) showed reactivities in ELISA assay using NP-40 membrane extracts of 3-hr schistosomula as antigens and in indirect immunofluorescence assay (IIF) using live 3-hr schistosomula. Both the protein A-bound and unbound fractions possessed high levels (84% and 76%, respectively) of complement-mediated cytotoxicity against schistosomula in vitro. The IgG- and IgM-containing fractions each conferred passive protection (30% and 20%) against challenge infection, although at a lower level when compared to unfractionated VMS (42%). These data demonstrate that in the mouse model both IgG and IgM can recognize epitopes on the surface of schistosomula, mediate cytotoxicity in vitro, and provide passive protection in vivo. Similarly, the protein A-bound and unbound fractions of vaccinated rabbit serum (VRS) were also shown to be positive in ELISA and IIF. The IgG- and IgM-containing fractions each possessed high levels (95% and 85%, respectively) of complement-dependent cytotoxicity against schistosomula in vitro. In contrast to VMS fractions, the IgG fraction of VRS conferred a similar level (28%) of in vivo protection as unfractionated VRS when injected into mice no later than 6 days after challenge. Moreover, the IgG fraction of VRS was still able to provide passive protection to mice when given as late as 15 days postinfection, but failed to confer protection when injected at 24 or 35 days postinfection.(ABSTRACT TRUNCATED AT 250 WORDS)

Three major surface antigens of Schistosoma mansoni are linked to the membrane by glycosylphosphatidylinositol.

Schistosomula of Schistosoma mansoni were examined for the presence of glycosylphosphatidylinositol (GPI) anchored surface membrane Ag. Parasites were surface iodinated and cultured in the presence or absence of a crude phospholipase C (PLC) preparation or phosphatidylinositol-specific PLC (PIPLC). Culture supernatants were then analyzed: 1) by centrifugation to ascertain which molecules released from the surface were soluble or contained in membrane vesicles; 2) by immunoprecipitation with antibodies specific for the "cross-reacting determinant," an epitope revealed on some GPI-anchored proteins only after cleavage of the diacylglycerol from the protein by PIPLC, and 3) by immunoprecipitation with immune mouse sera to establish co-identity with previously described, immunologically relevant surface Ag. By using these techniques, schistosomula were shown to possess three GPI-anchored surface Ag of m.w. 38,000, 32,000 and 18,000 which are spontaneously released from the surface of schistosomula in association with membrane, but remain insoluble until cleaved by PIPLC. All three molecules were recognized by antibodies from mice vaccinated with irradiated cercariae and/or chronically infected mice. Moreover, the m.w. 38,000 component was recognized by a previously described protective mAb (E.1). A major developmental modification appears to occur in the expression of these molecules because, by the same techniques, no GPI-anchored surface Ag were detectable on 7-day-old lung stage parasites. The finding that these important parasite immunogens are GPI-anchored and released from the surface of the parasite in membrane vesicles may, in part, explain why they elicit strong immune responses capable of damaging the schistosomulum tegument.

Effects of irradiation and tunicamycin on the surface glycoproteins of Schistosoma mansoni.

The cercarial glycocalyx and schistosomulum surface contains a number of glycoproteins which are expressed in very variable amounts within a parasite population. Tunicamycin inhibits glycoprotein synthesis of schistosomula if the parasites are incubated for 24 hr with the drug (10 micrograms ml-1). An unexpected increase in lectin binding to the parasite surface was observed but no other changes were detected. Schistosomula treated in this way did not develop in the host past the lung stage. Ultraviolet irradiation (400 microW min cm-2) also inhibited glycoprotein synthesis. Synthesis of other proteins, and in particular heat shock proteins, were also inhibited. Sera from mice (NIH strain) infected with irradiated cercariae contained antibodies which bound to normal schistosomula with lower affinity than to irradiated parasites. This is evidence that irradiation modifies the surface and secreted glycoproteins of schistosomula, so they are processed in a different way to normal glycoproteins by the host's immune system. The effects of irradiation on heat shock protein synthesis may allow the parasite to release a variety of proteins and glycoproteins in abnormal conformations. This may explain the enhanced immunogenicity of irradiated cercariae.

Experimental animal models in vaccination against schistosomiasis

Contrary to previous expectations, innate resistance to a primary schistosome infection is mediated predominantly in the lungs of many laboratory rodents. In addition, the phenomenon of non-permissiveness seen in a sub population of 129 strain mice, is associated with worm relocation from the liver to the lungs and is facilitated by dramatic alteration to the lung and liver vasculature; lung located adult worms exhibit gut damage and are ultimately destroyed within eosinophil-rich inflammatory focal reactions. It is now clear that the immunity induced by exposure to radiation-attenuated cercariae can be affected in the skin (mice), the lungs (mice and rats) or the liver (guinea pigs) of laboratory rodents. Moreover, the fact that skin phase resistances involves radio-sensitive cells, while lung and liver phase immunity centres on radio-resistant leucocytes, resolves current discord in the literature. Immobilisation and trapping of challenge larvae within focal inflammatory infiltrates is nevertheless common to both skin and lung phase attrition. Hyperimmunisation of rodents with irradiated cercariae is associated with a switch in immunoglobulin isotype and serum harvested from such donors is able to protect naive recipients passively; transferred serum recruits effector cells. Challenge parasites exhibit a broader window of sensitivity to vaccine immunity than was originally envisaged; stages ranging from the 3 to 4 day old skin/lung stage larva to the 3 week old juvenile liver worm constitute targets of protective resistance in vivo. This is at variance with the fact that newly transformed schistosomula constituting the primary targets of in vitro effector mechanisms, a feature perhaps related to our inability to mimic the process of intravascular parasite immobilisation and trapping in the test tube. Finally, schistosomicidal drugs such as Praziquantel can, by re-exposing disguised parasite antigens, facilitate the expression of vaccine immunity in sites additional to those at which resistance is normally mediated.

Antibody responses of rodents to a tegument membrane preparation from adult Schistosoma mansoni.

A preparation highly enriched in tegument membranes from adult worms has been used to probe rodent responses to schistosome surface antigens. All exposure regimens in mice stimulated levels of anti-tegument IgG, measurable by ELISA. Plateau levels of circulating antibody attained were similar after mixed-sex and male-only infections, and vaccination with tegument membranes. Female-only infections and exposure to irradiated cercariae stimulated lower levels of anti-tegument antibody. Responses in rats were similar, but occurred more rapidly, probably due to the large initial parasite exposure. Although both mice and rats could be stimulated to produce high levels of anti-tegument antibody by multiple vaccination, consistent protection against a challenge was not obtained. Paradoxically, rats and mice vaccinated with irradiated cercariae developed low levels of antibody to tegument membrane antigens but were highly resistant to challenge. It was concluded that, in the range of sera examined, antibody responses to schistosome tegument membranes did not correlate with the immune status of the host.

The regulation of resistance to Schistosoma mansoni by auto-anti-idiotypic immunity.

These studies explore auto-anti-idiotypic mechanisms as potential regulators of the protective immune response against Schistosoma mansoni. Anti-idiotypic responses were stimulated by immunization of mice with lymphoblasts, bearing specific idiotypic receptors. These receptors were produced in vitro by stimulation of Ag-reactive T cells by soluble cercarial immunogen, keyhole limpet hemocyanin, or Con A. The animals were then exposed to irradiated cercariae, keyhole limpet hemocyanin, or SRBC. The results indicate that the soluble cercarial immunogen lymphoblast recipient mice demonstrated reduction in a number of parameters of their immune response to schistosome Ag, including resistance to challenge by parasites. These changes were immunologically specific. Anti-idiotypic antibodies and anti-clonotypic T cell reactivity was demonstrated in the lymphoblast immunized mice. The suppression of reactivity in LBM was mediated by Lyt-1-, L3T-4-, and Lyt-2+ lymphocytes. These studies suggest that idiotypically dependent pathways might be important for the regulation of resistance to schistosomiasis.

Antigen localization and the induction of resistance in mice vaccinated with irradiated cercariae of Schistosoma mansoni.

The fate of 75Se-labelled parasites and their released pre-synthesized macromolecules has been followed in three murine infection models. Parasite numbers in specific tissues were determined by autoradiography, and released material was estimated by gamma-counting of tissues, with adjustment for the presence of parasite-associated radiolabel. Marked differences were found between the three models. The pattern of migration of normal schistosomula was similar to that previously reported. In addition we have described the transit of parasites through the lymph nodes draining the infection site. Significant quantities of released material were detected in the skin, draining lymph nodes, bloodstream and liver. The circulating material was of parasite origin, macromolecular, and hence potentially antigenic. In comparison to the normal infection, radiation-attenuated parasites (inducing a high level of resistance to challenge) persisted in the skin, draining lymph nodes and lungs, releasing a proportionally greater amount of material in the nodes. In mice exposed to attenuated parasites and treated with the compound RO11-3128 at 24 h (inducing a low level of resistance) there was an early death and rapid clearance of the parasites whilst still in the skin. This situation resulted in the highest levels of released material in the skin, bloodstream and liver, but negligible levels in the draining lymph nodes. We suggest that the persistence of radiation-attenuated parasites in the skin and draining lymph nodes, together with the prolonged release of antigen in the latter site, compared to the normal situation, are major factors in the induction of resistance.

Pulmonary leukocytic responses are linked to the acquired immunity of mice vaccinated with irradiated cercariae of Schistosoma mansoni.

Pulmonary cellular responses in C57BL/6 mice exposed to Schistosoma mansoni have been investigated by sampling cells from the respiratory airways with bronchoalveolar lavage. Mice exposed to cercariae attenuated with 20 krad gamma-radiation developed stronger and more persistent pulmonary leukocytic responses than animals exposed to equal numbers of normal parasites. Although vaccination with irradiated cercariae also stimulated T cell responses of greater magnitude and duration than normal infection, the lymphocytic infiltrate elicited by each regimen did not differ substantially in its composition, 5 wk after exposure. Studies with cercariae attenuated by different treatments established that a link exists between the recruitment of leukocytes to the lungs of vaccinated mice and resistance to reinfection. There was a strong association between pulmonary leukocytic responses and the elimination of challenge infections by vaccinated mice. Animals exposed to irradiated cercariae of S. mansoni were resistant to homologous challenge infection but were not protected against Schistosoma margrebowiei. Homologous challenge of vaccinated mice stimulated anamnestic leukocytic and T lymphocytic responses in the lungs, 2 wk postinfection, but exposure of immunized animals to the heterologous species failed to trigger an expansion in these populations of cells. Our studies indicate that pulmonary leukocytes and T lymphocytes are intimately involved in the mechanism of vaccine-induced resistance to S. mansoni. It remains unclear whether these populations of cells initiate protective inflammatory reactions against challenge parasites in the lungs, or accumulate in response to the activation of the protective mechanism by other means.

Analysis of the anti-Schistosoma mansoni surface antibody response during murine infection and its potential contribution to protective immunity.

Abstract Absorption of serum from chronically infected mice with homogenized schistosome eggs reduced antibody binding to the schistosomulum surface by 94%, indicating that almost all schistosomulum surface recognition during chronic infection is due to epitopes shared with the egg. Absorption of the serum with egg homogenate from which protein antigens had been removed by boiling and digestion with proteinase K resulted in a similar reduction of antisurface antibody demonstrating that all the shared epitopes that are recognized are carbohydrate in nature. Analysis of the time course of anticarbohydrate antibody production and the levels of antibody in mice infected with a single sex of schistosome indicated that eggs directly stimulated this response. Mouse mAb were identified that bound at very high levels to the schistosomulum surface and that recognized carbohydrate epitopes shared with the egg. Three of these had previously been demonstrated to passively transfer resistance, indicating that these surface carbohydrates are potential targets of protective immunity in the mouse. All the anticarbohydrate mAb also bound to the surface of schistosomula of other schistosome species. Thus, the strong immune response against these epitopes in chronic infection could account for the cross-specific immunity observed. Mice vaccinated with irradiated cercariae lacked high levels of anticarbohydrate antibodies and their recognition of the surface was largely due to antibody to species-specific polypeptide epitopes. With respect to the Mr greater than 200,000 and 38,000 antigens, it was demonstrated that these epitopes were present on the same antigens that bear the carbohydrate moieties recognized by antibodies from chronically infected mice. This specific polypeptide recognition is also reflected in the immunity generated by exposure to irradiated cercariae.

Examination of the mechanisms of pulmonary phase resistance to Schistosoma mansoni in vaccinated mice.

Potential mechanisms causing elimination of lung stage schistosomula were investigated in normal C57BL/6 mice and C57BL/6 mice previously vaccinated with irradiated cercariae. Autoradiography was used to measure the proportion of radiolabeled schistosomula which could be recovered from lung tissue by mincing and incubation. There was no difference between the two sets of mice at each time point, and no substantial diminution in the proportion recovered over the sampling period of 7 to 17 days post-infection. By this in vitro criterion, the emergence of schistosomula from lung fragments was not influenced by inflammation. However, we are unable to rule out the possibility that migration is impeded by inflammation in vivo. The migratory potential and viability of schistosomula recovered after residence in the lungs for increasing lengths of time was assessed by their introduction into the vasculature of naive recipients. The results indicate that significant numbers of schistosomula are capable of maturation, but fail to mature if left in situ in the lungs of both normal and vaccinated mice. No evidence was found that schistosomula suffer cytotoxic injury in the skin, subsequently dying in the lungs, or that they are damaged by the pulmonary inflammatory reactions which develop in vaccinated mice. Schistosomula delivered to the alveoli have a limited capacity to reenter tissues and mature. We suggest that in normal C57BL/6 mice the deflection of parasites into the alveoli during migration is the reason why many fail to mature. In vaccinated C57BL/6 mice a similar but augmented process may account for the elimination of a greater proportion of challenge parasites.

In vivo effects of monoclonal anti-L3T4 antibody on immune responsiveness of mice infected with Schistosoma mansoni. Reduction of irradiated cercariae-induced resistance.

Mice can be partially protected against challenge infections of Schistosoma mansoni cercariae by either single or multiple exposure to irradiated cercariae (x-cerc). The participation of L3T4+ lymphocytes on this resistance phenomenon was evaluated by selectively depleting this cell population through in vivo administration of mAb anti-L3T4 (hybridoma GK 1.5) at three different times in relationship to the challenge infections. Treatment with anti-L3T4 before challenge such that depletion was effective during the time of cercarial skin penetration and dermal/s.c. residence significantly reduced the level of resistance induced by x-cerc sensitization. When treatment was delayed until after challenge, depletion of L3T4+ cells coincided with either the lung or post-lung/liver phases of schistosomular migration, and normal levels of x-cerc-induced resistance were induced. In contrast to once-immunized mice, mice hyperimmunized by five exposures to x-cerc and then depleted of L3T4+ cells at the time of challenge (skin phase) still expressed resistance to the challenge. These data suggest that when mice are sensitized only once with x-cerc the challenge infection provides a necessary immunologic boost which requires L3T4+ cells for effective expression of resistance. The requirement for this anamnestic effect by the challenge infection can be circumvented by hyperimmunization. Evaluation of the immune response of one-time sensitized or hyperimmunized mice demonstrated that cellular Ag-specific proliferative responses and mitogen-induced lymphokine production were abrogated after any of the various in vivo regimens of anti-L3T4 antibody. In contrast, immunoblot analysis of humoral responsiveness revealed a correlation between the expression of resistance and the ability of sera from immunized and anti-L3T4 treated mice to recognize a 75-kDa parasite antigenic component.

Characterization of Schistosoma mansoni monoclonal antibodies which block in‐vitro killing: failure to demonstrate blockage of immunity in vivo

We have characterized various anti-schistosomular surface MoAbs previously shown to partially block in-vitro killing of schistosomula by human sera and eosinophils (Dunne et al. 1987). Immunodiffusion analysis showed that four IgM and one IgG3 MoAbs recognized periodate sensitive epitopes on the same molecular species present in schistosomular antigen but their patterns of reactivity with soluble egg antigen demonstrated that at least three distinct epitopes were involved. SDS-PAGE analysis showed the IgMs to react with 125I-labelled surface antigens of Mr 35,000-38,000 and Mr 20,000, and the IgG3 to react with an Mr 38,000 antigen. In spite of their effect in vitro, transfer of the IgM MoAbs at the time of challenge of mice vaccinated with irradiated cercariae or of mice injected with an unrelated (anti-Mr 16,000) protective MoAb failed to produce in-vivo blocking. Similarly, injection of Schistosoma mansoni eggs, prior infection with worms of one sex, or passive transfer of serum from single-sex infected mice failed to influence the resistance conferred by vaccination with irradiated cercariae.

Purification and immunochemical characterization of a 22 kilodalton surface antigen from Schistosoma mansoni

A 22 kDa antigen (Sm22) was purified from schistosomula membrane extracts by immunoaffinity chromatography with monoclonal antibody M.2. Western blotting suggested that the epitope bound by M.2 required a specific conformational folding of the molecule, which was sensitive to reducing agents. Two-dimensional electrophoresis of purified Sm22 demonstrated that the single 22 kDa protein recognized by M.2 on one-dimensional gel analysis was composed of at least two isomorphs. Additional Western blotting showed that Sm2 was one of the major antigens recognized by mouse anti-irradiated cercariac serum, and that this same serum recognized at least one epitope which was not sensitive to reducing agents. The mice vaccinated with irradiated cercariae were shown to be 75% protected from cercarial challenge. Sera from a rabbit immunized with Sm22 contained antibodies which bound to the surface of schistosomula and detected a single protein at 22 kDa by immunoprecipitation or Western blot. The rabbit anti-Sm22 sera also immunoprecipitated a 22 kDa in vitro translation product, indicating that at least one epitope on Sm22 is not dependent on glycosylation.

Genetic complementation of defects in vaccine-induced immunity against Schistosoma mansoni in P- and A-strain inbred mice

Inbred P- and A-strain mice are deficient in their capacity to develop resistance to challenge infection in response to vaccination with irradiated cercariae of Schistosoma mansoni and are also defective in their cell-mediated response as assessed by the activity of antigen-elicited macrophages in killing schistosome larvae in vitro. In contrast, vaccinated (P x A)F1 mice displayed high levels of both immunity to challenge and macrophage larvicidal activity, indicating that the P- and A-strain defects in the vaccine-induced response are controlled by distinct genetic loci.

Analysis of the anti-schistosoma mansoni surface antibody response during murine infection and its potential contribution to protective immunity

Absorption of serum from chronically infected mice with homogenized schistosome eggs reduced antibody binding to the schistosomulum surface by 94%, indicating that almost all schistosomulum surface recognition during chronic infection is due to epitopes shared with the egg. Absorption of the serum with egg homogenate from which protein antigens had been removed by boiling and digestion with proteinase K resulted in a similar reduction of antisurface antibody demonstrating that all the shared epitopes that are recognized are carbohydrate in nature. Analysis of the time course of anticarbohydrate antibody production and the levels of antibody in mice infected with a single sex of schistosome indicated that eggs directly stimulated this response. Mouse mAb were identified that bound at very high levels to the schistosomulum surface and that recognized carbohydrate epitopes shared with the egg. Three of these had previously been demonstrated to passively transfer resistance, indicating that these surface carbohydrates are potential targets of protective immunity in the mouse. All the anticarbohydrate mAb also bound to the surface of schistosomula of other schistosome species. Thus, the strong immune response against these epitopes in chronic infection could account for the cross-specific immunity observed. Mice vaccinated with irradiated cercariae lacked high levels of anticarbohydrate antibodies and their recognition of the surface was largely due to antibody to species-specific polypeptide epitopes. With respect to the Mr greater than 200,000 and 38,000 antigens, it was demonstrated that these epitopes were present on the same antigens that bear the carbohydrate moieties recognized by antibodies from chronically infected mice. This specific polypeptide recognition is also reflected in the immunity generated by exposure to irradiated cercariae.

Protective monoclonal antibodies from mice vaccinated or chronically infected with Schistosoma mansoni that recognize the same antigens.

An IgM monoclonal antibody, designated mAb 1.G1, has been generated from spleen cells of mice immunized with irradiated Schistosoma mansoni cercariae. As determined by indirect immunofluorescence, mAb 1.G1 binds to the surface membrane of schistosomula and to the ciliated plates of miracidia. mAb 1.G1 also binds to the protonephridial systems of live adult worms and denuded, acetone-fixed schistosomula. Western blot analysis shows that the target epitope of this mAb is found on Nonidet P-40-solubilized schistosomular antigens ranging in molecular size from 85 to 130 kDa and ciliated plate antigens of miracidia at 92, 95, and 102 kDa. The recognized epitope in an 8 M urea adult worm extract is found on a 97-kDa molecule. In addition, mAb 1.G1 mediates a high level of complement-dependent cytotoxic activity against schistosomula when used in an in vitro assay. In passive immunization experiments, approximately 40% protection was provided mice when mAb 1.G1 was administered either at the time of challenge or when given 8 days postchallenge. However, when administered 15 days postchallenge, mAb 1.G1 failed to mediate passive protection. The ability of mAb 1.G1 to mediate protection in vivo correlates with its recognition of epitopes on the surfaces of live schistosomula up to 8 days but not at 15 days. Western blot analysis showed that the antigens were contained within Nonidet P-40 extracts of schistosomula during the same time period. Furthermore, a second monoclonal antibody (mAb 4.4B) derived from mice chronically infected with S. mansoni exhibits the identical properties as described for mAb 1.G1.

Protective effect of irradiated metacercariae of Fasciola gigantica and irradiated cercariae of Schistosoma bovis against fascioliasis in goats.

Sensitisation of goats for eight weeks with metacercariae of Fasciola gigantica gamma-irradiated at 3 kr resulted in significant resistance to an homologous challenge with normal metacercariae. However, serum sorbitol dehydrogenase assay suggested that, whereas little damage was produced by immunising infections, considerable hepatic damage was caused by flukes remaining from challenge infections. On the other hand sensitisation with cercariae of Schistosoma bovis gamma-irradiated at 3 kr did not stimulate any resistance against heterologous challenge with F. gigantica.

Protein and antigen phosphorylation in the tegument of Schistosoma mansoni

Incubation of male and female Schistosoma mansoni for 3–6 h in media containing [ 32P]phosphate indicated that a large number of proteins extending across a wide molecular weight range were phosphorylated. The nature of the phosphorylated proteins was investigated by using three methods for removal of tegumental membranes. A 94 kDa polypeptide in the tegument became rapidly phosphorylated in vivo and it was also phosphorylated by incubation of isolated tegumental membranes in [ 32P]ATP in the presence of a protein kinase. After incubation of parasites in vivo for longer periods, other phosphorylated polypeptides of 62, 60, 57, 32 and 28 kDa were also identified. The major phosphorylated polypeptide immunoprecipitated from schistosomes by antisera raised in mice to irradiated cercariae was 62 kDa; others, of 48, 43 and 32 kDa, were also identified, using antisera raised in mice chronically infected with cercariae. The results suggest that mechanisms for receptor-mediated transmembrane signalling occur in the tegument of schistosomes.