Immunization of mice with gamma-irradiated intramuscularly injected schistosomula of Schistosoma mansoni

The parameters involved in the induction of resistance against Schistosoma mansoni by injection of irradiated, artificially transformed schistosomula were studied in mice. Single intramuscular injections of 500 schistosomula exposed to radiation doses in the range 2.3 to 160 krad. resulted in significant protection (in the range 20 to 50% as assessed by reduced worm burdens) against a challenge infection administered at intervals from 3 to 24 weeks post-vaccination. However, schistosomula irradiated with 20 krad. consistently resulted in better protection than those exposed to either higher or lower radiation doses despite the persistence of stunted adults from the infections irradiated with 2.3 krad. Vaccination with 40 krad. schistosomula resulted in significant protection in terms of reduced worm and tissue egg burdens and increased survival following lethal challenge. Varying the number of irradiated schistosomula, the frequency and route of their administration, the site of challenge and the strain of host all failed to enhance the level of resistance. However, percutaneously applied, irradiated cercariae were found to be more effective in stimulating resistance (60%) than intramuscularly injected, irradiated schistosomula (40%).     

The in vitro effects of various lysosomotropic agents on the gut of Schistosoma mansoni schistosomula

Cultured Schistosoma mansoni schistosomula of various ages were exposed to several lysosomotropic agents. Weak bases such as chloroquine, ammonium chloride, and acridine orange caused gut swelling upon protonation. The latter compound fluoresced a bright orange indicating the acidic nature of the gut contents. Hydrolysis of ingested L-amino acid methyl esters also resulted in gut swelling, indicating the nonpermeant nature of the resulting L-amino acids. Neither age nor feeding status influenced these swelling effects. Treatment of schistosomula with D-amino acid esters, free L-amino acids, or methanol had no effect. Thin-layer chromatographic analysis of worms treated with radiolabeled L-leucine methyl ester provided evidence that the ester was hydrolyzed. These results support the premise that the schistosome gut is an acidic compartment and is reminiscent of a secondary lysosome in its reaction to lysosomotropic agents.     

Peroxidase-mediated toxicity to schistosomula of Schistosoma mansoni

Guinea pig eosinophil peroxidase (EPO) was capable of killing schistosomula of Schistosoma mansoni in vitro when combined with hydrogen peroxide and a halide. Killing was measured by 51Cr release, by microscopic evaluation of viability, and by reinfection experiments in mice. Parasite killing was dependent on each component of the EPO-H2O2-halide system, was completely inhibited by catalase and azide, and was partially inhibited by cyanide. The EPO-mediated system required 10(-4) M H2O2 and 10(-4) M iodide at pH 7.0, and the schistosomula were killed with exposure to this system of less than 30 min at 37 degrees C. At pH 6.0, the EPO-mediated system showed significant cidal activity with 10(-6) M iodide. Canine neutrophil peroxidase (myeloperoxidase [MPO]) was also able to kill schistosomula in vitro in the presence of 10(-4) M H2O2 and 10(-4) iodide at pH 7.0 and pH 6.0. Physiologic concentrations of chloride (0.1 M) could substitute for iodide at pH 7.0 and pH 6.0 as the halide cofactor; however, at pH 7.0, a higher concentration of enzyme was required. These findings with isolated enzyme systems are compatible with a role for peroxidase in the host defense against schistosomula.     

Effect of praziquantel on the migration and survival of developmental stages of Schistosoma mansoni in mice

Compressed organ autoradiography has been used to determine whether the anthelmintic drug praziquantel (Pzq) modifies the migration of isotopically labelled Schistosoma mansoni during the first 16 days of infection in CBA/Ca mice. The mice were treated with 500 mg kg-1 body weight of the drug on day 1 or day 6. Treatment caused a marked delay in parasite migration from the skin when the drug was administered intradermally at the site of infection on day 1; migration from the lungs was also delayed after such treatment. Pzq injected either intradermally on day 1 or intramuscularly on day 6 effectively reduced the number of parasites that finally arrived in the lungs and the livers by 41 and 47%, respectively. Intramuscular administration of the drug on day 1 had a negligible effect. Worm recoveries assessed on day 38 by perfusion of the hepatic portal system were greatly reduced when Pzq was administered on day 14. The worms proved less susceptible when the drug was administered on day 21 and were completely resistant following drug delivery on day 28. The influence of drug preparation and route of delivery on parasite migration and survival are discussed.     

Pathology associated with vaccination against Schistosoma mansoni in mice using cryopreserved radiation-attenuated schistosomula

Twenty-one mice were injected intramuscularly with 2000 Schistosoma mansoni schistosomula irradiated at 20 krad and cryopreserved; three mice were killed on each of days 0, 2, 5, 9, 19, 28 and 44 days after infection and muscle from the site of injection in the left hind leg, the lungs and livers removed for histological examination. Schistosomula were seen in sections from the leg muscle from days 0 to 19 inclusive, in the lungs from day 2 to day 28 inclusive and in the livers from days 9 to 28 inclusive. Most schistosomula were seen in sections of the leg muscle with considerably fewer parasites occurring in the lungs and especially the livers. Granulomatous reactions comprising eosinophils, polymorphs, plasma cells and macrophages were first seen in the leg muscle on day 2, in the lungs on day 5 and in the liver on day 19. The peak inflammatory reactions appeared to occur between days 5 and 9, 9 and 19 and 28 and 44 respectively in the three tissues. The pathology is discussed in relation to the dose of irradiation required to attenuate the schistosomula for optimal immunogenicity.     

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.     

Schistosoma mansoni: effects on tryptophan metabolism in mice

In mice, infection with 20-30 cercariae of Schistosoma mansoni resulted in a considerable reduction in the formation of 14CO2 from [14C]tryptophan. Infected animals excreted significantly lower amounts of kynurenine, kynurenic acid, and methyl pyridone carboxamide than did uninfected controls. There was no difference in the ability of hepatocytes isolated from infected or control animals to metabolise [14C]tryptophan. Hepatocytes from infected animals synthesized less NAD(P), but more niacin and N1-methyl nicotinamide from tryptophan. They showed no greater accumulation of kynurenine metabolites than did cells from control animals. The hepatocyte content of pyridoxal phosphate and the erythrocyte aspartate aminotransferase activation coefficient were the same in both groups of mice, suggesting that infection with S. mansoni does not deplete vitamin B6. The impairment of tryptophan metabolism in vivo was apparently not due to impaired hepatic metabolism. Rather, it seems likely that the parasites or their eggs take up tryptophan avidly from the host's circulation. Studies of parasite and egg metabolism of tryptophan may suggest novel approaches to the chemotherapy of bilharzia.     

Schistosoma mansoni: long-term culture of in vitro derived schistosomula

In vitro derived schistosomula were cultured under various conditions. Variables tested included concentration of organisms, type of culture vessel, frequency of media change, time of erythrocyte addition, antibiotic levels, heated vs unheated serum in the media, and fresh vs stored serum or erythrocytes. No differences were observed between cultures changed every 3 or every 7 days, but worm growth and development were retarded when the culture medium was changed every 2 weeks. Organisms cultured without changing the medium for 3 weeks did not survive. High levels of antibiotics also inhibited growth and resulted in increased mortality. None of the other factors tested resulted in remarkable differences between groups. Some cultures lived for as long as 69 days, and pairing of adult worms was observed as early as 55 days. Most of the cultures, however, were lost before that time from the outgrowth of contaminants which were probably present in the cultures from the outset.     

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.     

The modulation of expression of polypeptide surface antigens on developing schistosomula of Schistosoma mansoni

Five low m.w. polypeptide antigens are expressed on the surface of freshly transformed schistosomula of Schistosoma mansoni, and were reproducibly identified by surface labeling with 125I by using IODOGEN and immunoprecipitating with immune mouse sera. These molecules have approximate m.w. of 38,000, 32,000, 20,000, 17,000, and 15,000. They correspond to antigens recognized previously by lactoperoxidase-catalyzed iodination. Analysis of the surface of developing schistosomulum demonstrated that the 38,000 and 17,000 dalton antigens were lost from the parasite surface during 48 hr of in vitro culture. This process was not dependent on the presence of host serum. The two antigens were not lost due to shedding into the culture medium but were apparently sequestered to a site where they were no longer available for surface labeling. The 32,000, 20,000, and 15,000 dalton antigens, however, remained exposed on the schistosomulum surface for up to 2 days of in vitro culture. The expression of two new antigens was also induced by culture in vitro: a doublet of approximately 45,000 daltons and an antigen of approximately 11,000 daltons. The expression of the former was dependent on the presence of serum. These results demonstrate that the development of the schistosomula surface is a complex process, with events both dependent and independent of the presence of serum. In addition, the expression of polypeptide antigens is not coordinated, and antigens are lost, retained, or appear on the schistosomulum surface during the early stages of maturation.     

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: expression and role of cysteine proteinases in developing schistosomula

The adult stage of Schistosoma mansoni utilizes host hemoglobin as a nutrient source. A proteolytic enzyme (SMw32) that has "hemoglobinase" activity is secreted into the parasite gut where it appears to be rapidly activated by glutathione released from host red blood cells. In the present study the expression of this proteinase, in developing schistosomula, has been correlated with digestive tract development and a dramatic rise in enzyme activity as early as Days 8-10 of culture. No evidence of the SMw32 proteinase was found in eggs, cercariae, or in newly transformed larvae. Further, the proteinase expressed at Days 8-10 is indistinguishable from the adult worm enzyme. In the larvae, indirect immunofluorescence with an anti-SMw32 monoclonal antibody showed that the proteinase is found throughout the developing cecum. The importance of cysteine proteinases to parasite development was also studied using a specific enzyme inhibitor, Ep-459. In cultures containing Ep-459 most (75%) of the schistosomula failed to survive the 18-day study period. Moreover, those that did survive showed a decrease in their growth (body length). These data suggest that the SMw32 proteinase is a developmentally regulated enzyme and that cysteine proteinase activity is essential in providing nutrients for the growth and survival of this parasite in its mammalian host. Thus, this proteinase may be an important target for chemotherapeutic intervention.     

A comparative study of the death of schistosomula of Schistosoma haematobium and Schistosoma mansoni in the skin of mice and hamsters.

This study shows that some cercariae of S. haematobium and S. mansoni die during penetration of mouse or hamster skin. Approximately 30-38% of cercariae of both species die in mouse skin and 14-16% die in hamster skin. The greater number of cercariae which die in the skin of mice seems to account for the higher yield of adult worms recovered in hamsters. Adult worm recoveries from animals infected with S. haematobium were, however, only about half the worm recoveries from hosts infected with S. mansoni.