Schistosoma mansoni: increasing saline concentration signals cercariae to transform to schistosomula

Cercariae of S. mansoni shed the surface glycocalyx, form a double lipid bilayer on their surface, and transform to schistosomula when tails are removed and parasites are transferred from pond water to 300 mOsm phosphate-buffered saline. To determine whether the absolute concentration of saline or the relative change in saline concentration was the signal for surface transformation, cercariae were isolated from the snail hepatopancreas, sheared to remove the tails, and incubated in defined media for 3 hr at 37 degrees C. Surface transformation was assayed using the binding of the fluorescein-conjugated lectin concanavalin A to the schistosomular double unit membrane but not to the cercarial glycocalyx. An increase in salinity either from 18 mOsm (artificial pond water) to 120 mOsm (the snail osmolarity) or from 120 to 300 mOsm (the mammalian osmolarity) triggered transformation to schistosomula. Organisms constantly exposed to 120 mOsm or shifted from 120 mOsm to pond water did not transform their surfaces. The signal for transformation appeared to be increasing salinity rather than increasing osmolarity because cercarial bodies did not become schistosomula in 300 mOsm mannitol. Surface transformation was inhibited when cercariae were incubated with the acetylcholinesterase inhibitor eserine sulfate during a 10 min time when the osmolarity was raised. We conclude that increasing salinity rather than the absolute saline concentration is the signal for surface transformation and that eserine sulfate may inhibit the receipt of this signal.     

The cercarial glycocalyx of Schistosoma mansoni

Cercariae, the freshwater stage of Schistosoma mansoni infectious to man, are covered by a single unit membrane and an immunogenic glycocalyx. When cercariae penetrate the host skin, they transform to schistosomula by shedding tails, secreting mucous and enzymes, and forming microvilli over their surface. Here the loss of the glycocalyx from cercariae transforming in vitro was studied morphologically and biochemically. By scanning electron microscopy, the glycocalyx was a dense mesh composed of 15-30 nm fibrils that obscured spines on the cercarial surface. The glycocalyx was absent on organisms fixed without osmium and was partially lost when parasites aggregated in their own secretions before fixation. By transmission electron microscopy, a 1-2 microns thick mesh of 8-15-nm fibrils was seen on parasites incubated with anti-schistosomal antibodies or fixed in aldehydes containing tannic acid or ruthenium red. Cercariae transformed to schistosomula when tails were removed mechanically and parasites were incubated in saline. Within 5 min of transformation, organisms synchronously formed microvilli which elongated to 3-5 microns by 20 min and then were shed. However, considerable fibrillar material remained adherent to the double unit membrane surface of schistosomula. For biochemical labeling, parasites were treated with eserine sulfate, which blocked cercarial swimming, secretion, infectivity, and transformation to schistosomula. Material labeled by periodate oxidation and NaB3H4 was on the surface as shown by autoradiography and had an apparent molecular weight of greater than 10(6) by chromatography. Periodate-NaB3H4 glycocalyx had an isoelectric point of 5.0 +/- 0.4 and was precipitable with anti-schistosomal antibodies. More than 60% of the radiolabeled glycocalyx was released into the medium by transforming parasites in 3 h and was recovered as high molecular weight material. Parasites labeled with periodate and fluorescein-thiosemicarbazide and then transformed had a corona of fluorescence containing microvilli, much of which was shed onto the slide. Material on cercariae labeled by lodogen-catalyzed iodination was also of high molecular weight and was antigenic. In conclusion, the cercarial glycocalyx appears to be composed of acidic high molecular weight fibrils which are antigenic and incompletely cleared during transformation.     

Schistosoma mansoni and Biomphalaria glabrata have some common epitopes. I. Common epitopes are present on the surface of early stages of S. mansoni.

1. The presence of snail and glycocalyx antigens in the Schistosoma mansoni cercarial surface and their permanence throughout development in vitro and in vivo was investigated. 2. Rabbit antisera raised against two fractions of glycocalyx released from cercariae and Biomphalaria glabrata soft tissues or haemolymph were obtained. 3. All four antisera bound to cercariae and schistosomula kept in vitro or in vivo for up to 24 hr. 4. No binding to schistosomula kept in vivo for 5 days or longer was observed. 5. Schistosomula cultured in vitro for up to 12 days bound the antisera throughout the period of culture.     

Schistosoma mansoni: in vitro conversion of cercariae to schistosomula.

Schistosoma mansoni cercariae were incubated for 3-5h at 37 degrees C in various test solutions, and the rate and extent of conversion of the cercariae to schistosomula were determined. Criteria used to identify schistosomula included: (1) loss of cercarial tail, (2) viability of organisms in saline but not in water, (3) pre-acetabular gland evacuation and (4) ability to survive in culture. Incubation of cercariae in rat chamber fluid resulted in organisms which were water sensitive, but retained their tails and pre-acetabular gland contents. Conversion to water sensitivity was not blocked by 0-01 M EDTA.     

Schistosoma mansoni: immunogenic glycoproteins of the cercarial glycocalyx

Immunochemical studies at the level of the light and electron microscope showed that a monoclonal antibody, 128C3/3, was directed to an epitope in the glycocalyx of Schistosoma mansoni cercariae. Immunoprecipitation of surface labeled cercarial extracts with this monoclonal antibody demonstrated that the glycocalyx is composed of at least five components, including a very large molecular size polypeptide and polypeptides of 220, 180, 170, and 15 kDa. After transformation of cercariae to schistosomula, these polypeptides were shed from the surface and were therefore no longer accessible to surface labeling. Monoclonal antibody 128C3/3 was also reactive with a 38 kDa polypeptide from schistosomula; this polypeptide was weakly expressed on the surface of cercariae. Analysis of immunoprecipitates of radioiodinated protein extracts of cercariae, newly transformed schistosomula, and 36 hr in vitro cultured schistosomula showed that the 180 and 170 kDa polypeptides continued to be expressed within the organism following transformation, but were not accessible to surface labeling. Lectin binding studies revealed differences in the oligosaccharide composition of the six polypeptides. With the exception of the 15 kDa antigen, all the polypeptides reactive with 128C3/3 were highly immunogenic in infected mice and humans.     

Ultrastructure, carbohydrate, and amino acid analysis of two preparations of the cercarial glycocalyx of Schistosoma mansoni

This study determined the amino acid and carbohydrate composition of 2 cercarial glycocalyx preparations obtained after phenol-water extraction and subsequent gel chromatography. Labeled cercariae were subjected to 85% phenol, thereby dissociating the glycocalyx into the aqueous phase, which was dialyzed and chromatographed on Sepharose CL-2B or -4B in either 4 M guanidine hydrochloride (GuHCl) or 0.1% sodium dodecyl sulfate (SDS). The label eluted primarily in the void volume and was antigenic as tested with rabbit polyclonal antibodies by immunoblotting. Approximately 6 micrograms of protein and 28 micrograms of carbohydrate were obtained from 10(5) cercariae in the antigenic void volume fraction after SDS chromatography. Threonine, serine, and glutamic acid comprised 44% of the amino acid residues of the protein. The predominant sugar was fucose. Galactosamine, glucosamine, galactose, and mannose were also detected. After GuHCl chromatography, free amino acids, predominantly glycine and serine, comprised 17% of the total protein. The carbohydrate composition was similar to that of SDS-chromatographed extracts. Phenol-water extracts eluting in the void volume of Sepharose CL-2B were compared by negative staining and scanning electron microscopy with material obtained from medium in which cercariae shed glycocalyx during transformation to schistosomula. Both the isolated material and the transformation medium contained particles 20-50 nm in diameter, with subunits of 15-20 nm. These studies show that the cercarial glycocalyx is particulate, contains mainly carbohydrate and some protein, and is solubilized by phenol-water extraction.     

Schistosoma mansoni: topochemical features of cercariae, schistosomula, and adults

The teguments of developing and mature cercariae, recently transformed, and 1-wk-old schistosomula and adult worms were examined for the ultrastructural location of macromolecular carbohydrates and polyelectrolytes. The surface of mature cercariae within sporocysts and cercariae released from the snail is covered by a filamentous coat which reacts with cytochemical reagents for the demonstration of vicinal glycols, but neither the coat nor the surface of the tegument plasmalemma binds cationic colloidal iron at low pH.Upon penetrating mammalian skin, the cercaria sheds its surface coat; the tegument surface of newly transformed schistosomula, older schistosomula and adult worms stains en bloc with acidic colloidal iron, as does the tegument plasmalemma of mature cercariae if the overlying filamentous coat is first removed by physicochemical means. The cercarial coat thus serves to mask anionic groups at the surface of the tegument plasmalemma which become functionally exposed after penetration of the mammalian host. The distribution of colloidal iron binding sites coincides with those for the carbohydrate-complexing phytohemmagglutnin, concanavalin A, which suggests that these membrane-fixed anions are acid mucopolysaccharides, glycoproteins or glycolipids. Carbohydrate-containing material was also localized within membrane-bound vesicles of the tegument matrix and perikarya of developing cercariae and postcercarial schistosomes, suggesting that surface mucosubstances contributing to the tegument glycocalyx of these worms are elaborated, at least in part, by the tegument itself.     

Surface proteins on schistosomula and cercariae of Schistosoma mansoni

The surface proteins of schistosomula and cercariae of Schistosoma mansoni were examined by lactoperoxidase catalysed iodination followed by polyacrylamide gel electrophoresis. Two polypeptide chains were clearly labelled and they were present on both cercarial and schistosomular surfaces. A number of minor labelled components were also observed. The results suggest that membranes of the teguments of cercariae and schistosomula are similar.     

Schistosoma mansoni: protein phosphorylation during transformation of cercariae to schistosomula.

Infectivity of the multicellular pathogen Schistosoma mansoni for the human host is dependent upon the ability of free-living cercariae to transform rapidly into parasitic schistosomula. The biochemical pathways that regulate this transitional period are unknown. The role of protein phosphorylation was investigated by examining the incorporation of [32Pi]phosphate into proteins of S. mansoni. A sevenfold increase in total phosphorylation was found in 3-hr-old schistosomula as compared to cercariae. Analysis of radiolabeled proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated that a 14-kDa protein served as a marker for transformation, being phosphorylated in schistosomula but not cercariae. The protein was phosphorylated on a serine residue. Phosphorylation was stimulated by a shift of parasites from water to salt-containing medium at 23 degrees C. Incubation of organisms in water at 37 degrees C did not initiate phosphorylation of this protein. The 14-kDa phosphoprotein was extracted from parasite homogenates with 1 M NaCl but was insoluble in 1% Triton X-100. Protein phosphorylation during the cercarial-schistosomula transformation may represent an important biochemical event that regulates infectivity of the parasite for the human host.     

Evidence that a protective membrane epitope is involved in early but not late phase immunity in Schistosoma mansoni

An anti-egg monoclonal antibody E.1, which is partially protective in passive transfer experiments, is shown in this study to recognize a membrane epitope on cercariae, schistosomula, and the ciliary plates of miracidia. E.1 did not bind to the surface membranes of lung or adult worms, or recognize secreted egg antigen in infected liver tissue. The E.1 epitope was present in the glycocalyx of cercariae, as well as on the syncytial membrane as determined by electron microscopy. Immunoprecipitation of iodinated surfaces of cercariae and schistosomula demonstrated E.1 binding to a high m.w. moiety in cercariae, which corresponds to the glycocalyx because it was not immunoprecipitated from schistosomula. In addition, a band at 38,000 daltons was immunoprecipitated from both cercariae and schistosomula. When compared with in vitro cultured parasites, schistosomula that were obtained from mice 1 to 24 hr after tail vein injection showed significant loss of E.1 binding. Consistent with the rapid loss of antigen in vivo, E.1 antibody was unable to passively transfer protection to naive mice when administered 5 days after cercarial challenge.     

<Emphasis Type="Italic">Trichobilharzia regenti</Emphasis>: Antigenic structures of intravertebrate stages

Like several other bird schistosomes, neurotropic schistosome of Trichobilharzia regenti can invade also mammals, including humans. Repeated infections cause cercarial dermatitis, a skin inflammatory reaction leading to parasite elimination in non-specific mammalian hosts. However, in experimentally primo-infected mice, the worms escape from the skin and migrate to the central nervous system. In order to evade host immune reactions, schistosomes undergo cercaria/schistosomulum transformation accompanied with changes of surface antigens. The present study is focused on localization of the main antigens of T. regenti; cercariae, schistosomula developed under different conditions and adults were compared. Antigens were localized by immunofluorescence and ultrastructural immunocytochemistry using sera of mice repeatedly infected with T. regenti. Detected antibody targets were located in glycocalyx and penetration glands of cercariae and in tegument of cercariae, schistosomula and adults. Shedding of cercarial glycocalyx significantly reduced surface reactivity; further decrease was reported during ongoing development of schistosomula. Spherical bodies, probably transported from subtegumental cell bodies to worm surface, were identified as the most reactive tegumental structures. Based on similar results for schistosomula developed in specific, non-specific hosts and in vitro, it seems that the ability of T. regenti to decrease the surface immunoreactivity during ontogenesis is independent on the host type.     

Schistosoma japonicum (Chinese): Changes of the tegument surface in cercariae, schistosomula and juvenile parasites during development

, , , and 1988. Schistosoma japonicum (Chinese): changes of the tegument surface in cercariae, schistosomula and juvenile parasites during development. International Journal for Parasitology18: 1093–1104. The surface of cercariae of S. japonicum (Chinese) and changes following their transformation into schistosomula were observed at 0.5,1, 3,6,12,24 and 48 hand 5,7,10 and 15 days by scanning electron microscopy. The cercarial surface shows two distinct regions: on the head and the body it is mildly undulating and covered throughout with numerous spines that are partially embedded in a thick layer of glycocalyx; on the tail it is highly folded into tall and thin circumferential ridges, spines are confined to the dorsal and ventral aspects and are prominent due to a thinner glycocalyx. Between 0.5 and 12 h following skin penetration, the surface of schistosomula is gradually cleared of glycocalyx, while small blebs and slender microvilli project out from the glycocalyx-free area and from spines. Subsequently, these structures are detached from the surface and thus responsible for the shedding of the original cercarial membrane and glycocalyx. Between 24 and 48 h, schistosomula show little change in the body size in comparison to earlier stages, and most of the surface is still covered with spines but devoid of blebs and microvilli. From day 2 onwards schistosomula are lengthened but with little net growth. Spines decrease in the middle region of the body; and the surface is invaginated by small pits and folded into thin ridges, which result in rapid increase of the surface area. Between days 5 and 7 schistosomula elongate further, and the disappearance of the spines in the middle region becomes even more pronounced, while those remaining are confined only to the anterior and posterior extremities. Ridges and pits are more developed, and the former begin to show regular foldings throughout all parts of the body. Between days 7 and 10 schistosomula grow rapidly and the body is quadrupled in size. Between days 10 and 15 sexes can be differentiated. The male has a flat ovoid shape body, whose ridges and pitting on the surface are highly developed almost to the same extent as in adult parasites, while spines remain mostly on the posterior end. Sensory papillae greatly increase in number, especially on the ventral and lateral aspects and around the suckers. The surface of the female is relatively smooth; ridges are developed only on the posterior part, while the remaining surface is flat and extensively pitted. Spines are present throughout but less concentrated in the middle region.     

Schistosoma mansoni: development of the cercarial glycocalyx

The development of the cercarial glycocalyx of Schistosoma mansoni was studied by transmission electron microscopy and immunofluorescence light microscopy employing antibodies raised against extracted and chromatographed glycocalyx. By electron microscopy, cercariae present in the brood chamber of daughter sporocysts were surrounded by an electron-dense granular and fibrillar matrix. This material appeared structurally distinct from the glycocalyx which was coarsely fibrillar and located only on the surface of organisms that had developed a final tegument. The thickness of the glycocalyx apparently increased with the maturation of the tegument, since teguments that had many spines also had the thickest glycocalyx. Immunofluorescent staining of frozen sections of infected snail hepatopancreas showed that glycocalyx antigens were present on the surface of the cercariae and not in the matrix within the brood chamber or in snail tissues. Immunofluorescent staining of isolated larval cercariae showed staining of some but not all parasites with partially elongated tails. These studies suggest that the glycocalyx develops late in cercarial development (late in Stage 6 or in Stage 7 of Cheng and Bier), is made by the cercariae themselves, and is not a product of either the sporocyst wall cells or snail hepatopancreas.     

The presence of antibody in mice chronically infected with Schistosoma mansoni which blocks in vitro killing of schistosomula

We have previously reported that IgM monoclonal antibodies (mAb) that recognize surface carbohydrate determinants shared between schistosomula, cercariae, and miracidia block antibody/complement dependent killing of schistosomula in vitro. Binding assays that make use of one of the IgM mAb labeled with 125I demonstrated that serum from chronically infected mice (CMS) contained high levels of competing antibody, whereas serum from mice vaccinated with irradiated cercariae (VMS) contained little antibody of this specificity. Absorption of CMS with cercariae that removed antibodies to schistosomulum surface carbohydrate determinants increased its ability to kill schistosomula in vitro; absorption of VMS with cercariae failed to alter the lethal activity of the serum. Furthermore, fractionation of CMS by protein A Sepharose chromatography demonstrated that the IgG fraction had an increased lethal activity compared with unfractionated serum; this result was not seen with VMS. Finally, the IgM fraction of CMS was shown to block in vitro killing of the IgG fractions of both CMS and VMS. These data suggest that the blocking activities observed with the IgM mAb are contained within the serum of chronically infected mice but not in the serum of mice vaccinated with irradiated cercariae.     

Reappraisal of experimental infections with cercariae and schistosomula of a Brazilian strain of Schistosoma mansoni in mice.

The present investigation involves a reevaluation of previous results obtained after experimental infection of Swiss Webster mice with cercariae and schistosomula of the Schistosoma mansoni LE strain maintained under laboratory conditions. Three experimental groups of mice were considered: the animals of the first group were percutaneously (ring method) infected with cercariae, those of the second were subcutaneously inoculated with cercariae and the mice of the third were inoculated by the same route with schistosomula transformed in vitro. The data obtained so far indicated that the most effective method of infection is the subcutaneous injection with schistosomula, with a mean adult worm burden recovery of 54.1% when compared to the abdominal percutaneous and subcutaneous routes of infection with cercariae, in which the values were 36.7% and 32.4%, respectively. This suggests that, in experimental infections of SW mice with a LE S. mansoni strain, the skin is to be considered an effective attrition site in the percutaneous route, whereas in the case of inoculation with cercariae, a small amount of larvae fails to be transformed into viable schistosomula, possibly due to skin phase avoidance. A brief discussion about attrition sites and elimination of larval S. mansoni worms in mice is presented.     

Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay

A radioimmunoassay was developed to screen supernatants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosoma mansoni. Of 196 clones screened, 10% bound schistosomula. Of these, 74% bound only schistosomula. The remaining molecules also reacted with soluble adult worm antigens and soluble egg antigens as determined by enzyme-linked immunosorbent assay. Immunoblot analysis demonstrated that monoclonal antibody 204-3E4 reacted with a 68 kDa protein, a glycoprotein that induces substantial resistance against S. mansoni infection. Recognition of an 18 kDa antigen by 204-3F1 antibody was stage-specific with the antigen being expressed in cercariae, 3- and 24-h-old parasites but not 4-day, lung stage or adult worms. Monoclonal antibody 204-4E3 reacted with purified S. mansoni paramyosin. These data indicate that radioimmunoassay using living schistosomula is a rapid alternative method to identify murine hybridomas that secrete antibodies which react with surface antigens of S. mansoni.     

Schistosoma mansoni: sterol and phospholipid composition of cercariae, schistosomula, and adults

The sterol and phospholipid composition of cercariae, schistosomula, and adult Schistosoma mansoni was analyzed by gas-liquid chromatography and high-performance liquid chromatography (HPLC). Cercariae and schistosomula contained cholesterol, desmosterol, campesterol, stigmasterol, and beta-sitosterol while adults contained only cholesterol. In all stages cholesterol comprised greater than 50% of the total sterols, and in cercariae and schistosomula desmosterol comprised 38 and 21% of the total sterols, respectively. The other three sterols, campesterol, stigmasterol, and beta-sitosterol, made up approximately 10% of the total. The same five sterols found in cercariae and schistosomula were present in the hepatopancreas of uninfected snails but with a much higher desmosterol concentration in the parasite, 38%, than in the snail, 2%. As in cercariae and schistosomula the three minor sterols comprised approximately 10%. Thus, the sterol composition of cercariae and schistosomula was similar but not identical to that of the snail host. Phosphatidylcholine was the major phospholipid of all three stages (50%) as determined by two HPLC procedures. The remaining phospholipids consisted of phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. In addition, in adults there were small quantities of sphingomyelin and lysophosphatidylcholine. The percentage of each phospholipid was similar among stages with the exception of a slight increase in phosphatidylserine in adults compared to cercariae and schistosomula. These results show that a characteristic lipid composition is found in cercariae, schistosomula, and adults.     

Adoptive transfer of protective immunity in experimental schistosomiasis in the mouse

Passive transfer of immune serum alone did not confer protection to recipient mice irrespective of the routes of serum transfer or cercarial challenge of Schistosoma mansoni. Mice that received both sensitized cells and immune serum were protected against challenge by subcutaneous injection of cercariae but not by percutaneous exposure. The immune serum could be transferred as late as 8 days after subcutaneous challenge, suggesting that the protection was afforded in part by a late parasite killing mechanism which functions after the schistosomula have migrated through the lungs.     

Role of eosinophils in the destruction of schistosomula of Schistosoma mansoni in vivo (preliminary report)

Schistosoma mansoni cercariae mechanically transformed into schistosomula were injected, either dead or alive, into the tail vein (2.000 larvae/0,15 ml) of Balb/c mice which were either previously infected with S. mansoni (10 weeks/50 cercariae) or non-infected. Histological examination of the lungs 24, 48, 72 and 96 hours after injection revealed that inflammatory reaction around schistosomula occurred only in the groups injected with dead schistosomula (killed by freezing and thawing). In non-infected animals the reaction was predominantly macrophagic while in those infected many eosinophils appeared around the dead larvae. These results are at variance with those obtained in vitro and suggest that in vivo the participation of eosinophils in the schistosomulum induced reaction in sensitized animals is secondary to the death of the larvae.     

Oxygen uptake and lactate production by Schistosoma mansoni cercaria, cercarial body and tail, and schistosomule.

1. Oxygen consumption by Schistosoma mansoni cercarial bodies varies, with the batch of organisms, the incubation media and the temperature (27-37 degrees C), from 27.4 +/- 3.4 to 55.0 +/- 4.8 microliters O2/mg larval protein per hr. It is proportional to the concentration of organisms incubated, up to 25,000/ml, as calculated from whole protein. 2. Oxygen uptake by cercariae is inhibited by 5.6 mM glucose in the incubation media, a concentration that stimulates the respiration of cercarial bodies. 3. No significant differences in the oxygen uptake were presented by cercarial bodies with and without glycocalyx or glandular secretions, or devoid of all of them. 4. Inhibitors of the Krebs cycle and the respiratory chain, and uncoupling agents influence the oxygen uptake by cercariae, cercarial bodies and schistosomules to the same extent. 5. The permeability change presented by transformed larvae had no influence on the excretion of lactate by cercarial bodies, which is about 0.3 mumoles/mg protein per hr and remains constant for 5 hr; under nitrogen, this amount increased 70%. Cercariae in anaerobiosis, however, excreted as much as 15 times more lactate than under air. 6. Lactic dehydrogenases of cercariae, cercarial bodies and tails, and schistosomules are of the muscle type and do not change during the transformation.