Schistosoma mansoni: detection by monoclonal antibody of a 22,000-dalton surface membrane antigen which may be blocked by host molecules on lung stage parasites

Monoclonal antibody M.2 binds to the surface membranes of cercariae and developing schistosomula. This antibody was generated from mice immunized with membrane-enriched extracts of mechanically transformed schistosomula. The antigen detected by M.2 was shown to persist on developing schistosomula for at least 96 hr post-transformation. M.2 also bound to the surface of living, cultured lung worms but not to freshly harvested lung worms. The ability of M.2 to bind to cultured lung worms coincided with the loss of host H-2 from the parasite surface. The apparent m.w. of the antigen was 22,000; an antigen with the same apparent m.w. was immunoprecipitated from cercariae, schistosomula, lung worms, and adult worms.     

Schistosoma mansoni: rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll

Step gradients of polyvinylpyrolidone-coated colloidal silica particles (Percoll) were used to isolate and purify early development stages of Schistosoma mansoni (cercariae, skin stage, and 5-day-old schistosomula). With this method, mechanically transformed schistosomula can be isolated in higher purity and yield than that obtained with conventional procedures. In addition, use of the method revealed that schistosomula undergo a dramatic change in density during the first hours after transformation from cercariae. In other experiments, 5-day-old schistosomula were effectively purified from contaminating lung tissue by means of the Percoll gradient procedure. After purification on Percoll, schistosomula display no evidence of damage when examined by light microscopy and no loss in viability as judged by recovery of adult worms from mice.     

Schistosoma mansoni: molecular characterization of Alkaline Phosphatase and expression patterns across life cycle stages

Here we describe the cloning and characterization of the Schistosoma mansoni Alkaline Phosphatase (SmAP), previously identified in the tegument of adult worms. SmAP encodes a complete sequence composed of 536 amino acids containing an N-terminal signal peptide, five N-glycosylation sites, and a GPI anchor signal, similar to that described for mammalian orthologs. Real-time RT-PCR and Western blot experiments suggest a rapid translation as soon as cercariae are transformed into schistosomula. Immunolocalization analysis shows that the protein is widely distributed in the worm tissues, with increased concentration in the vitelline glands of female parasites. Furthermore, the surface localization of this enzyme was quantitatively supported by its enzymatic activity in live ex vivo or cultured parasites throughout the life cycle stages. The fact that cercariae accumulate large amounts of SmAP mRNA, which rapidly translates into protein upon schistosomula transformation, indicates it may have an important role in host invasion.     

Surface antigens on cercariae,schistosomula and adult worms of Schistosoma mansoni

Shah J. and Ramasamy R. 1982. Surface antigens on cercariae, schistosomula and adult worms of Schistosoma mansoni. International Journal for Parasitology12: 451–461. The surface protein antigens of Schistosoma mansoni were radiolabelled by lactoperoxidase catalysed I¹²⁵-iodination and analysed by immune-precipitation and polyacrylamide gel electrophoresis. The results showed that regularly labelled surface antigens of mol. wts >150,000, 78,000, 45,000 and 22,000 were present on adult worms. Common surface antigens were observed on the cercariae, schistosomula and adult worms. It is suggested that surface antigens released from living adult worms can sensitise a host to react against the invading schistosomula of a secondary infection. However, the failure to vaccinate mice using material containing adult worm surface antigens suggests that the induction of protective immunity is a complex phenomenon.     

Expression of lectin-binding surface glycoproteins during the development of Schistosoma mansoni schistosomula

Tegumental glycoproteins of Schistosoma mansoni cercariae, mechanically produced 24-hr and 48-hr schistosomula, and adult worms were radioiodinated with the Bolton-Hunter reagent, then isolated by lectin affinity chromatography. SDS-PAGE revealed Con A binding glycoproteins with apparent molecular weights of 180,000, 150,000, 43,000, and 30,000 in detergent extracts of the tegument of cercariae. These glycoproteins are retained by 24-hr mechanically produced, cultured schistosomula and are accompanied by the appearance of 2 additional labeled glycoproteins, mol. wt. 66,000 and 57,000. In 48-hr schistosomula, there is a marked increase in the relative size of the 66,000 mol. wt. peak. In contrast, the 57,000 mol. wt. glycoprotein is the major radiolabeled Con A binding component of the adult tegument; the other peaks are either reduced or absent in adults. Similar findings were obtained following affinity chromatography using immobilized Lens culinaris lectin or Ricinus communis agglutinin, and following metabolic labeling of glycoproteins with tritiated galactose.     

<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 mansoni Annexin 2: Molecular characterization and immunolocalization

We here describe the cloning and characterization of the Schistosoma mansoni Annexin 2, previously identified in the tegument by proteomic studies, and as an up-regulated gene in schistosomulum stage by microarray data. In silico analysis predicts a conserved core containing four repeat domains of Annexin (ANX) and a variable N-terminal region similar to that described for mammalian isoforms. Real-time RT-PCR and Western blot analysis determined that S. mansoni Annexin 2 is significantly up-regulated in the transition from free-living cercaria to schistosomulum and adult worm parasitic stages. Immunolocalization experiments and tegument membrane preparations confirmed Annexin 2 as a protein mainly localized in the tegument of schistosomula and adult worms. Furthermore, it binds to the tegument surface membranes in a calcium-dependent manner. These results suggest that S. mansoni Annexin 2 is closely associated to the tegument arrangement, being a potential target for immune intervention.     

Schistosoma mansoni host-exposed surface antigens characterized by sera and recombinant antibodies from schistosomiasis-resistant rats

Antibodies from Schistosoma mansoni-infected rats, unlike mice, show a higher titer for schistosome apical tegumental antigens compared with non-apical membrane antigens. These antibodies bind to the surface of living lung-stage worms and to formaldehyde-fixed adult worms. We produced a single-chain antibody Fv domain (scFv) phage library displaying the antibody repertoire of rats highly immune to schistosome infection and we selected for scFvs that recognize the host-exposed surface of worms. Five unique rat scFvs (Teg1, Teg4, Teg5, Teg20 and Teg37) were obtained which recognize schistosome surface epitopes. Each of the scFvs recognizes the surface of living schistosomula and lung-stage schistosomules and/or the surface of formaldehyde-fixed adult worms. None of these scFvs reproducibly stained living adult worms. This suggests that a change occurs during the transition from lung schistosomules to 4-week adults such that at least some surface antigens, although remaining on the surface in living adult worms, can no longer be immunologically stained. Teg1 and Teg4 scFvs both recognize specific bands on Western blots. No bands were observed for the other three scFvs, suggesting that these scFvs may recognize non-protein or conformationally-dependent epitopes. Teg1 was unambiguously identified as recognizing the S. mansoni tetraspanin antigen, SmTSP-2, within the large extracellular domain. Teg4 recognizes a 35 kDa band tentatively identified as Sm29 by proteomic analysis. These scFvs can now be used to characterize schistosome epitopes at the host-parasite interface, to target worms in vivo, and to study the mechanisms by which these worms naturally evade immune damage to the tegument within permissive hosts.     

日本血吸虫尾蚴及童虫的生理学比较

本文介绍了经0.05％中性红-氢氧化钾染色确定为活的血吸虫尾蚴和童虫的生理学比较结果。与尾蚴相比,童虫体表对PAS及Alcian蓝阳性反应消失,与抗血清接触不再产生套膜反应;对水不能耐受,在淡水中6小时内已全部死亡,而在生理盐水中仍有92.7％存活;钻腺内含物排空,童虫不能再经皮肤感染,但越过皮肤屏障若被注入皮下或腹腔则分别有45.4—46.5％及56.9—64.9％发育为成虫。     

Schistosoma mansoni: Tegumental damage as a consequence of lectin binding

Adult worms of Schistosoma mansoni exhibit gross tegumental damage following incubation in concanavalin A or Ricinus communis agglutinin. However, incubation with wheat germ agglutinin induces only minimal surface damage, while soybean agglutinin has no damaging effect upon the worms. Damage induced by Ricinus communis agglutinin or concanavalin A may be prevented by the addition of the appropriate competing sugar. In contrast, incubation of 3-hr artificially transformed schistosomula in concanavalin A and other lectins does not produce any disruption of the tegument. These results indicate that the surface membrane of the adult schistosome is readily disrupted by ligand binding and appears to be particularly sensitive and fragile. The membrane of the schistosomulum, however, is more resistant to the effects of lectin binding. Adult worms incubated in culture medium alone (ELAC or RPMI 1640) show background changes which seem to be related to the tonicity of the medium. Such results advocate that preliminary assessment of schistosome integrity be carried out prior to any experimental procedures which preclude the addition of serum to the basic incubation medium. Schistosomula do not exhibit comparable sensitivity.     

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.     

β-Lapachone: A naphthoquinone with promising antischistosomal properties in mice

The activity of β-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione, β-lap) against different stages of Schistosoma mansoni was investigated in mice. Mice infected with 50 cercariae (BH strain) were intraperitoneally treated at a dose of 50 mg/kg for 5 consecutive days, starting on the 1st, 14th, 28th and 45th days after infection, to evaluate the effect of β-lap on skin schistosomula, lung schistosomula, young worms (before oviposition) and adult worms (after oviposition), respectively. All animals were euthanized 60 days after infection. β-Lap significantly reduced (p < 0.001) the number of worms in 29.78%, 37.2%, 24.2% and 40.22% when administered during the phases of skin schistosomula, lung schistosomula, young worms and adult worms, respectively. Significant reduction was also achieved in terms of female burden. In all groups, there was significant reduction in the number of eggs and granulomas in the hepatic tissue. When the intervention was performed during the phase of adult worms, β-lap reduced the size of hepatic granulomas and changed the oogram pattern, lowering the percentage of immature eggs and increasing the percentage of mature and dead eggs. Our data indicate that β-lap has moderate antischistosomal properties. Its molecule may also be used as a prototype for synthesis of new naphthoquinone derivatives with potential schistosomicidal properties. Further studies with different formulations containing β-lap are needed to clearly establish the best dose and route of administration and its mechanism of action against schistosomes.     

Scanning electron microscopical studies on the tegument of adult worms of Schistosoma mansoni originating from ultraviolet-irradiated and non-irradiated cercariae.

The surface topography and ultrastructural changes of Schistosoma mansoni worms developed from ultraviolet-irradiated cercariae were compared with those from non-irradiated cercariae. The tegument of worms developed from irradiated cercariae showed a variety of changes including the occurrence of oedemata and most of the tubercles being torn at the tip and having lost their spines on the dorsal surface of some male worms. In females, parts of the tegument were devoid of spines and surface lesions were present. The range and extent of these changes differed not only among individual worms but also between different regions of individual worms resulting in generalized deformities in those worms originating from irradiated cercariae.     

Two isoforms of a divalent metal transporter (DMT1) in Schistosoma mansoni suggest a surface-associated pathway for iron absorption in schistosomes

We describe two homologues of the mammalian divalent metal transporter (DMT1) for Schistosoma mansoni, a pathogenic intravascular parasite of humans. Schistosomes have a high nutritional and metabolic demand for iron. Nucleotide sequences of the parasite homologues, designated SmDMT1A and -B, are identical in all but the 5'-regions. The predicted amino acid sequences share at least 60% identity with DMT1 (=Nramp2) of humans, mice, and rats, and at least 55% identity with Nramp1 from mice, humans and Caenorhabditis elegans. SmDMT1A is expressed in differentiating eggs, miracidia, cercariae, schistosomula, and adults, whereas SmDMT1B is expressed in all but the miracidium and occurs at lower levels than SmDMT1A in differentiating eggs and cercariae. An iron-responsive element, present at the 3'-untranslated region of many DMT1 molecules, is not present in schistosome mRNAs studied here. A Western blot analysis of adult worm preparations using a homologous rabbit serum raised against a schistosome DMT1 peptide and a heterologous serum raised against mammalian DMT1, revealed a band approximating 115 kDa. By immunofluorescence microscopy, the schistosome DMT1s localize primarily to the tegument. Iron uptake assays demonstrated that SmDMT1s were able to rescue yeast growth in ferrous iron-transport deficient yeast (fet3fet4). The results suggest that schistosomes express molecules for ferrous iron transport in their tegument, suggesting trans-tegumental transport as one means of iron acquisition for these parasites.     

Schistosoma mansoni: identification and characterization of schistosomula polypeptides

Schistosomula proteins separated by a two-dimensional (NEPHGE) gel system identify 94 major silver-stained polypeptides. When compared to polypeptides similarly separated from cercariae and adult worms; cercariae share the same polypeptides as schistosomula, adult worms share ca. 60% of the polypeptides. A group of five schistosomula polypeptides 15-31 kDa (apparent pI 8.2-8.9) was not found in adult worm extracts. To identify which polypeptides were immunogens, Western blots of the NEPHGE gels were probed with sera either from humans with chronic schistosomiasis or from mice vaccinated with irradiated cercariae. For characterization studies, polyclonal antibodies were made against the five schistosomula-specific and selected immunogenic polypeptides by immunizing mice with silver-stained spots removed from NEPHGE gels. We show that the polyclonal serum against a polypeptide of 12.5 kDa and an apparent pI of 6.70 mediated complement and eosinophil-dependent killing of schistosomula in an in vitro assay. Epitopes recognized by antibody against the 12.5-kDa polypeptide show a diffuse distribution and are found on flame cells of the excretory system of the schistosomula.     

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.     

日本血吸虫在小鼠体内发育形态的扫描电镜观察

日本血吸虫童虫在移行和发育过程中体形发生了明显的演变,并形成了口吸盘和排泄孔,同时也伴随着许多细微结构的改变。这些改变包括:体褶和体嵴数量及高度的增加、体棘数目和位置的改变、感觉器数量和分布的变化。