Andreas Ruppel

Anthraquinones in Rheum palmatum and Rumex dentatus(Polygonaceae), and phorbol esters in Jatropha curcas(Euphorbiaceae) with molluscicidal activity against the schistosome vector snails Oncomelania,Biomphalaria, and Bulinus

Hot water extracts of Rheum palmatum and Rheum dentatus (from China) showed molluscicidal activity against the snails Oncomelania hupensis, Biomphalaria glabrata and Bulinus globosus which are vectors of Schistosoma japonicumS. mansoni and S. haematobium respectively. Activity was correlated with anthraquinones which were identified by HPLC: rhein and chrysophanol‐anthron were most active (>50% dead snails after 2 days in a 0.03% solution). Molluscicidal activity was intermediate with Rheum‐emodin and physcion and was not detectable with cinnamic acid or Aloe‐emodin. The snail O. hupensis tended to be more sensitive for several compounds than B. glabrata Extracts of Jatropha curcas seeds (from Mali) showed molluscicidal activity against both B. glabrata and O. hupensis the latter being the more sensitive snail. The activity was associated with phorbol esters extracted from Jatropha oil. Of the pure phorbol esters tested, 4β‐phorbol‐13‐decanoate killed both snail species at a concentration of 0.001% (10 p.p.m). As Jatropha is locally grown in Mali for other purposes, it might potentially be exploited for schistosomiasis control.

SmCB2, a novel tegumental cathepsin B from adult Schistosoma mansoni.

Papain-like cysteine endopeptidases have been recognized as potential targets for chemotherapy and serodiagnostic reagents in infections with the human parasitic helminth Schistosoma. A novel cathepsin B endopeptidase from adult S. mansoni has been isolated and characterized. The enzyme is termed SmCB2 to distinguish it from the first recorded schistosome cathepsin B, SmCB1, also known as Sm31. A rapid and convenient protocol involving anion exchange and affinity chromatography is described for the isolation of SmCB1 and SmCB2 from the same parasite starting material. SmCB2 has been functionally expressed in and purified from Pichia pastoris. Both native and recombinant SmCB2 migrate similarly (33 kDa) by SDS-PAGE. Both display strict acidic pH activity profiles and similar K(m) and k(cat) for dipeptidyl amidomethylcoumarin substrates. We conclude that the recombinant enzyme is properly folded. The S(2) subsite specificity of recombinant SmCB2 exhibits the preferences Phe>Leu>Val>>Arg. By immunoblotting with anti-SmCB2 IgG, a 33 kDa protein was identified in soluble extracts of male schistosomes. By immunohistochemistry, SmCB2 was localized in the tegumental tubercles and parenchyma of males with less product being visualized in the parenchyma of females. The enzyme may be lysosomal and function at the host parasite-interface.

Schistosoma japonicum: An ultraviolet-attenuated cercarial vaccine applicable in the field for water buffaloes

Water buffaloes were vaccinated three times with 10,000 Schistosoma japonicum cercariae irradiated with ultraviolet (uv) light at a dose of 400 microW x min/cm2. The irradiation was performed with cheap, simple, and portable equipment in a rural area of Hubei Province (Peoples Republic of China). A challenge infection of 1000 untreated cercariae was given to six vaccinated and six naive control buffaloes, while two vaccinated animals were not challenged. The experiment was terminated 6 weeks after the challenge. Control animals had lost body weight and harbored a mean of 110 worms and 37 eggs per gram of liver. The vaccinated animals gained weight after the challenge and developed 89% resistance to infection with S. japonicum. Since schistosomiasis japonica is nowadays transmitted in China predominantly by domestic livestock, a uv-attenuated cercarial vaccine for bovines may contribute to the control of this disease.

Schistosoma mansoni and S. japonicum : comparison of levels of ultraviolet irradiation for vaccination of mice with cercariae

When cercariae of Schistosoma mansoni and of S. japonicum were irradiated with various levels of u.v. light at 254 nm, their development to perfusable worms was reduced to below 1% at about 200 microW min cm-2. Cercariae attenuated with about 300 microW min cm-2 induced partial resistance against an homologous challenge infection in mice. No differences were observed between the two schistosome species when the same treatment was given to the cercariae. Thus the same u.v. dose can confer immunizing ability to cercariae of both S. mansoni and S. japonicum.

Cathepsin B-like activity predominates over cathepsin L-like activity in adult Schistosoma mansoni and S. japonicum

Abstract Both cathepsin B-like and cathepsin L-like endopeptidase activities have been described in schistosomes, but their relative contribution to proteinolysis remains controversial. In an attempt to clarify which type of activity predominates, the selective mammalian cathepsin B inhibitor CA-074 was tested under standardized assay conditions with different preparations from Schistosoma mansoni and S. japonicum. CA-074 (0.94 μM) inhibited at least 92% and 80% of proteinolytic activity, respectively, for these species: completely inhibited bovine-spleen cathepsin B activity; but showed only marginal inhibition (4%) of rat-liver cathepsin L activity. We discuss the results with respect to previous studies and conclude that schistosome cathepsin B-like, not L-like, activity predominates.

Schistosoma mansoni: Immunoblot analysis of adult worm proteins

Proteins of adult Schistosoma mansoni were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assayed in immunoblots for reactions with individual mouse sera. Four weeks after a heavy infection with a few hundred cercariae, IgG antibodies directed predominantly against a protein of 31 kDa were detected. The protein was only weakly recognized by antibodies of mice harboring a 4-week-old light infection with about 60 cercariae. After 6 weeks or more, mice infected with either dose formed antibodies, not only against the 31-kDa protein and a 67-kDa protein, but also against a number of other components. While reactions with the 31- and 67-kDa proteins occurred with sera of all individual mice of four different strains, the reactions with other components were less consistently observed. Mice vaccinated with a heavy or light dose of 20,000-rad-irradiated cercariae did not form antibodies detectable in the blotting system. However, in immunofluorescence assays with living skin schistosomula, but not lung schistosomula, antibodies against the larval surface were detected with all sera obtained 4 weeks after infection or vaccination. In addition, immunofluorescence studies using the same sera and sectioned adult parasites demonstrated the presence of antibodies against the parasite surface in all sera except those obtained from mice exposed to a light infection with normal cercariae. Mice infected in this latter way were the only animals that did not develop a significant resistance against a challenge infection 4 weeks after exposure to normal or irradiated cercariae. The presence of an immunofluorescent reaction against the schistosome gut always coincided with a reaction of the sera with the 31-kDa protein in the immunoblots. Although a role in immune resistance could not be ascribed to any of the proteins reacting in the immunoblots, the data demonstrate important differences in the antibody specificities induced by various infection schemes.

Cloning of diagnostic 31/32 kilodalton antigens of Schistosoma mansoni

A cDNA library derived from messenger RNA of adult Schistosoma mansoni was constructed in lambda gt11 and schistosome antigens were expressed as fusions with the amino terminus of the beta-galactosidase of Escherichia coli. Using mouse and human infection sera, recombinant clones specific for a 31/32 kDa doublet having a potential in the immunodiagnosis of schistosomiasis were selected. The specificity of the clones was verified by their reactivity with monoclonal antibodies. The identity of the cloned epitopes with those of the native proteins was confirmed by Western blot analysis of total schistosome proteins, using both antibodies affinity purified from the fusion proteins and antisera raised in rabbits against the fusions. The reactivity of the cloned antigens with human infection sera suggests their usefulness for the immunodiagnosis of human schistosomiasis.

DNA vaccination with asparaginyl endopeptidase (Sm32) from the parasite Schistosoma mansoni: anti-fecundity effect induced in mice.

DNA-based vaccine technology was used to induce an immune response in mice against a schistosome cysteine proteinase, asparaginyl endopeptidase (Sm32). The cDNA coding for Sm32 was cloned in a mammalian expression vector under control of the CMV promoter/enhancer and expressed for the first time in transfected mammalian cells as well as in mice immunized with the Sm32-encoding DNA construct. These mice developed antibodies which recognized the native protein not only in homogenates of Schistosoma mansoni worms but also in the gut on cryostat sections of the parasites. This DNA vaccine led to an anti-fecundity effect: female worms of a challenge infection produced 37% less eggs than those growing in naïve mice. The results suggest that Sm32 may be a candidate antigen for the generation of an anti-pathology vaccine against schistosomes.

Schistosoma haematobium, S. intercalatum, S. japonicum, S. mansoni, and S. rodhaini in mice: relationship between patterns of lung migration by schistosomula and perfusion recovery of adult worms.

Abstract The development of five schistosome species was compared in mice by the recovery of schistosomula from chopped lung tissue and of adult worms by portal perfusion. Three developmental patterns appeared. (1) Schistosoma japonicum was unique in showing an early establishment of schistosomula in and a rapid departure from the lungs together with the highest worm recovery; (2) S. haematobium contrasted by establishing later and persisting in the lungs for at least 2 weeks while yielding the lowest adult worm recovery; and (3) S. intercalatum, S. mansoni, and S. rodhaini had an intermediate pattern – they resided in the lungs for several days, then disappeared and produced intermediate numbers of adults. Lung petechiae, known to accompany the migration of S. japonicum, were never detected after infection with the other species. We speculate that the three migration patterns of schistosomes are related to the size of the relative spectra of naturally infected definitive hosts.

Schistosoma japonicum and S. mansoni: comparison of larval migration patterns in mice

Mice were infected percutaneously with cercariae of Schistosoma japonicum or S. mansoni and parasites recovered by tissue-mincing from the skin or lungs or by perfusion of the mesenteric veins. S. japonicum had a narrow peak of recovery (up to 30%) from the lungs 3 days after infection, whereas lung recovery of S. mansoni peaked only on day 6 and levelled off during the following week. Infection with S. japonicum induced lung petechiae, but only after most of the parasites had left the lungs. The axillary lymph nodes draining the infection site increased in weight after infection and this effect was much greater and longer with S. mansoni than with S. japonicum. S. japonicum was perfusable from the mesenteric veins earlier (from day 3 onwards) and in higher number (40-60% from days 6 to 10) than S. mansoni (20% on day 20). The percentage of cercariae developing to adult worms was 57% for S. japonicum and 33% for S. mansoni. The data demonstrate that S. japonicum might escape from local tissue reactions in the skin and lungs and, due to its rapid migration, might induce only poor lymphocyte proliferation. As a possible consequence, S. japonicum may establish more efficiently in mice than S. mansoni.