Schistosoma mansoni: Cloning by microsurgical transplantation of sporocysts

Development of a method of infecting of the molluscan host by microsurgical transplantation of the parasite's sporocysts enables the researcher to maintain the host cycle of Schistosoma mansoni exclusively by asexual means and without the participation of a vertebrate host. After transplantation, larval morphogenesis becomes altered to form an additional generation of sporocysts. These invade the digestive gland of the recipient mollusc progressively, producing normally infective cercariae. The maintenance of the life cycle of S. mansoni in the laboratory for 1 year, solely in the mollusc, has been obtained through six successive transplantations. Thus, a true cloning of S. mansoni has been achieved, the original transplant material being derived from a monomiracidial infection. From the practical viewpoint, this transplantation technique is of definite utility in the maintenance of the cycle, the vertebrate stage having been eliminated. From the theoretical viewpoint, unexpected analogies become apparent with the two types of larval demography found in Digenea (Digenea with sporocyst and Digenea with rediae).     

Schistosoma mansoni: Cytotoxicity of hemocytes from susceptible snail hosts for sporocysts in plasma from resistant Biomphalaria glabrata

Sporocysts of Schistosoma mansoni (PR1 strain) survive and grow in Biomphalaria glabrata PR albino strain snails, whereas they are encapsulated and die in B. glabrata 10R2 strain snails. These processes also occur in an in vitro system in which the only living cells are those of sporocysts and snail hemolymph. Hemocytes of the susceptible snail are normally not effective in damaging sporocysts. However, when the encounter occurred in the presence of cell-free plasma from resistant snails, previously impotent hemocytes severely damaged sporocysts in 24 hr. The cytotoxic capacity of resistant strain hemocytes was not altered by plasma from susceptible snails. Furthermore, it was retained even when plasma was replaced by culture medium free of snail components. The nature of the plasma factor(s) which facilitated damage by otherwise impotent hemocytes is discussed, and evidence is evaluated for the hypothesis that snail resistance is dependent upon the specificity of cytophilic factors present both in the plasma and on the hemocyte plasma membranes.     

Schistosoma mansoni: Lysozyme activity in Biomphalaria glabrata during infection with two strains

Levels of lysozyme activity were determined in the hemolymph, digestive gland, and headfoot extracts of M-line stock of snails, Biomphalaria glabrata, during infection with the PR-1 and Lc-1 strains of the trematode, Schistosoma mansoni. At 3 hr postexposure there was a 10-fold increase in the levels of enzyme activity in the hemolymph of snails infected with the Lc-1 strain to which the snail is resistant. This increase was considerably higher when compared to the threefold increase in the PR-1-infected snails. The infection also induced a gradual depletion of lysozyme activity in the headfoot muscles of the two groups of infected snails. There were no changes in the levels of enzyme activity in the digestive gland extracts of the control and the two groups of infected snails. Similar changes in the levels of enzyme activity in the hemolymph and headfoot extracts of infected snails suggest a nonspecific response to a parasite infection and do not indicate that lysozyme is primarily responsible for the destruction of schistosome parasite in a resistant snail host.     

Schistosoma mansoni: Vaccination of mice with highly X-irradiated cercariae

Vaccination against schistosomiasis with highly X-irradiated Schistosoma mansoni cercariae was studied in mice. The optimum dose of X radiation for the attenuation of cercariae was in the range of 24–48 krad. In selecting the optimum dose, lesions caused by migrating schistosomula in the lungs of the immunized host were considered. Cercariae exposed to 48 krad caused fewer lesions than those exposed to 24 krad but still effected a comparable worm reduction. The percentages of worm reduction in mice immunized with 48-krad X-irradiated cercariae increased with the number of immunizations up to the fifth immunization and then fluctuated in the sixth, seventh, and eighth days without increase. The optimum dose of immunizing cercariae was 500, and the optimum time interval for successive immunizations was 4 weeks. There was no significant difference in susceptibility to infection in the adult mice 161 to 694 days of age. The duration of acquired immunity in immunized mice is long, still evident 545 days from the last immunization. The present studies clearly showed that with the bioengineering method, the worm reduction in the immunized mice reached 91.1%, the effect of immunization was stronger in mice immunized with the highly X-irradiated cercariae than with the low X-irradiated cercariae, and X-irradiated cercariae were demonstrated to be a strong inducing agent for immunity in mice.     

Schistosoma mansoni: Agglutination of sporocysts,and formation of gels on miracidia transforming in plasma of Biomphalaria glabrata

The resistance or susceptibility of Biomphalaria glabrata strains to strains of Schistosoma mansoni, the human blood fluke, are evidenced by the responses of snail hemocytes to sporocysts of the schistosome, both in vivo and in vitro. It is now reported that living sporocysts of the PR1 strain of S. mansoni agglutinate in the plasma of all tested strains of B. glabrata, in contrast to fixed sporocysts which agglutinate only in plasma from resistant snail strains. The agglutinating activity in resistant plasmas is not divalent cation dependent, and was not inhibited by the 26 carbohydrates and four amino acids tested. In addition, the observation that gelatinous deposits develop on transforming miracidia-sporocysts in B. glabrata plasmas is also reported. Both the agglutination and gel-formation phenomena may facilitate recognition of, and attacks on, sporocysts, thereby contributing to susceptibility and resistance in this host-parasite system.     

Schistosoma mansoni and Schistosoma haematobium: Emergence of schistosome cercariae from snails with darkness and illumination

Biomphalaria glabrata and Bulinus globosus were infected with Schistosoma mansoni and Schistosoma haematobium, respectively, and the effect of different illumination conditions at 25 C on cercarial output was observed for 4 days. In both species, a dark period of 10–14 hr on Day 2 of the observation period resulted in an emergence pattern on Day 3 similar to the regular pattern recorded for Day 1. Total cercarial output on Day 3 was within 30% of the control (Day 1) output. A dark period of between 0 and 8 hr resulted in suppression of cercarial emergence and in abolishment of the regular hourly emergence pattern on Day 3. A dark period of 16–20 hr resulted in an emergence pattern with two peaks, the first occurred at Hour 1, and the other at Hour 5 of the subsequent light period. Interjection of a 1-hr dark period during the light period of Day 3, following short (2–8 hr) exposure to dark on the preceding day, produced an increase in cercarial shedding of S. mansoni immediately after restitution of the light conditions. On the other hand, in S. haematobium, cercarial output was stimulated during the interposed dark period itself.     

Schistosoma mansoni: Ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules

Against the background of cercarial fine structure, ultrastructural changes were compared in schistosomules of Schistosoma mansoni 30 min and 1 hr after their production in vivo by skin penetration and in vitro by shear pressure. The same developmental pattern was observed in schistosomules of both derivations. In vitro schistosomules, however, developed more slowly, resembled cercariae more closely, and varied less among organisms than did in vivo schistosomules. The greatest morphological changes were observed in the 1-hr in vivo schistosomules. These were as follows: (1) in tegument, formation of transient microvilli, a hepatalaminate outer membrane and accented surface invaginations, loss of glycocalyx, movement outward of cyton vesicles via bridges, accumulation of multilaminate bodies around bridge openings; (2) in the anterior organ (oral sucker), movement of head gland vesicles via the ducts into tegument followed by collapse of the gland fundus, disappearance of the circumfundal cells and two large support cells, and the appearance in these areas of membranes and parenchymal cells; (3) secretion of the acetabular gland contents, collapse of the glands and replacement by membranes and parenchymal cells; (4) peristaltic activity of the digestive tract as shown by alternate areas of lumen constriction and dilation; (5) loss of bladder and contraction of the small aboral collecting tubules; and (6) conversion of heterochromatic parenchymal cell nuclei to euchromatic. In contrast, the 1-hr in vitro shear schistosomules resembled 30-min in vivo schistosomules, retaining many cercarial features.     

Schistosoma haematobium: Amoscanate and adult worm ultrastructure

Amoscanate, when administered orally as an aqueous or “formulated” preparation, induced pronounced ultrastructural abnormalities in male and female Schistosoma haematobium. Higher dose levels of the aqueous suspension (300 mg/kg body wt) had to be administered to achieve the full range of effects induced by formulated doses of 2.5–8 mg/kg body wt. Worms were recovered from hamsters between 1 and 120 hr after treatment. Although the amount of amoscanate-induced damage varied considerably between worms, an overall pattern of damage emerged. Initially, 1 hr after treatment, amoscanate caused tegumental vacuolation and oedema. As the drug treatment period was extended to 24 hr, blebbing, exudation, collapse of sensory organelle bases, and abnormal mitochondria became increasingly evident. With exposure to higher drug doses (50–300 mg/kg body wt), the tegument became further distorted with the appearance of necrotic structures and myelin whorls, which appeared to represent various stages in lysosomal formation and digestion. Eventually, erosion of surface layers resulted in the breakdown of tegumental integrity. The caeca and vitellaria were also adversely affected by drug treatment. Basal vacuolation and the formation of myelin whorls occurred in the gastrodermis. In the mature S4 vitelline cells, coalesced vitelline droplets and myelin whorls were evident.     

Schistosoma mansoni: Complement activation by antigenic preparations

The ability of antigens prepared from adult worms and eggs of Schistosoma mansoni to activate complement in vitro in normal, human serum in the absence of specific antibodies was investigated. It was demonstrated that whole viable eggs activated the complement system; this was shown to be effected by egg antigens released into the medium. Egg-hatching fluid induced a high degree of complement consumption, whereas purified egg shells gave almost no complement consumption. The complement-activating antigens of the eggs are possibly of polysaccharide nature as indicated by an almost complete complement activation by trichloroacetic acid-soluble egg antigens. No detectable complement consumption occurred upon incubation of living adult worms, but antigens extracted from adult worms did give complement consumption. Circulating cathodic antigens and excretory and secretory antigens proved to be quite capable of inducing complement activation; tegumental antigens gave lower, but still significant levels of complement consumption.     

Schistosoma mansoni: characterization of clones maintained by the microsurgical transplantation of sporocysts

Five male and 5 female clones of Schistosoma mansoni were established and maintained for 3 yr by the serial microsurgical transplantation of sporocysts from infected to uninfected Biomphalaria glabrata snails. The clones were initially derived from 10 randomly selected snails with monomiracidial infections. Clones were characterized by several criteria, including their infectivities for mice and snails, their cercarial outputs, and their ability to produce immunity in mice. The mean infectivities of individual clones in mice ranged from 26 to 44%, and were highly consistent within each clone. The infectivities of cloned sporocysts in snails ranged from 44 to 100% and were also highly consistent within clones. Mean cercarial outputs from individual clones ranged from 450 to 4,300 per snail. In mice, clones differed significantly from each other in their ability to immunize and in their susceptibility to immunity. Each clone was unique and did not appear to differ with time or subpassaging through snails, suggesting that the differences had a genetic basis.     

Schistosoma mansoni: Tyrosine,a putative in vivo substrate of phenol oxidase

l-Tyrosine, l-[3,4]dihydroxyphenylalanine (l-DOPA), and dopamine are known to be in vitro substrates for Schistosoma mansoni phenol oxidase. Since all three compounds are present in the female schistosome, it is not clear which one serves as the substrate for phenol oxidase in intact S. mansoni. However, the concentration of l-tyrosine in the female schistosome (252 ng/mg worm) is 4-fold higher than the K_m of phenol oxidase for this amino acid while the concentrations of l-DOPA and dopamine (0.954 and 0.790 ng/mg worm, respectively) are 100- and 500-fold lower than the K_m of these substrates. Tri-l-tyrosine methyl ester is oxidized at less than 3% of the rate of l-tyrosine methyl ester. A tyrosine:lysine peptide and chymotrypsinogen are not oxidized. Female S. mansoni do not incorporate l-tyrosine into proteins to a significantly greater extent than l-leucine. The results suggest that free l-tyrosine is the substrate for S. mansoni phenol oxidase in vivo.     

Schistosoma mansoni: Purification and characterization of malate dehydrogenases

Isoelectric focusing of a homogenate of Schistosoma mansoni, followed by malate dehydrogenase-specific staining, showed the presence of two major and five minor malate dehydrogenase isoenzymes (EC 1.1.1.37), with isoelectric points ranging from 7.3 to 9.5. The malate dehydrogenase isoenzymes were purified by gel filtration, followed by ion-exchange chromatography on DEAE- and CM-cellulose. The isoenzymes could be differentiated by their susceptibility to substrate inhibition. No differences in the Michaelis-Menten constants for substrate were found. One of the isoenzymes is inhibited by 5′-AMP. Further purification of this particular isoenzyme was achieved by affinity chromatography on 5′-AMP-Sepharose 4B. Analysis after subcellular fractionation indicated a mitochondrial origin for this isoenzyme. The mitochondrial isoenzyme (at a recovery of 80%) was purified 218-fold compared to the crude soluble extract, and contained about 40% of the total malate dehydrogenase activity. The enzyme has a molecular weight of 65,500 and showed absolute specificity for l-malic acid, NAD, and NADH. The final preparation has a specific activity of 451 U/mg protein. Physicochemical studies, including binding constants, substrate inhibition, thermostability, and pH optima, demonstrated differences between the mitochondrial and cytoplasmic enzymes. A role for malate dehydrogenase in Schistosoma mansoni metabolism is discussed.     

Schistosoma mansoni: Inhibition of glucosephosphate isomerase and glycolysis by sugar phosphates

Glucosephosphate isomerase (EC 5.3.1.9) of Schistosoma mansoni is inhibited competitively by a number of tetrose, pentose, and hexose phosphates with inhibitor constant (K_i) values in the range of 0.5 to 400 μM. The most potent inhibitor is 5-phospho-d-arabinonate which resembles the cis-enediolate transition state intermediate of the reaction. These analogs were also found to be effective inhibitors of the production of lactate from glucose by suitably supplemented worm homogenates. The rank order of potency of inhibition of glycolysis was inversely related to the magnitudes of the K_i values for glucosephosphate isomerase. These K_i values were similar to those previously reported for mammalian glucosephosphate isomerase, suggesting similarities in the steric and electronic characteristics of the active sites of these isofunctional enzymes. This conclusion was further supported by the observed pH dependence of the inhibition by 5-phospho-d-arabinonate. Although glucosephosphate isomerase is not a rate-limiting enzyme of glycolysis, in the conventional sense, its selective inhibition could be of chemotherapeutic importance, in part because of the accumulation in glycolyzing systems of glucose 6-phosphate which is a potent feedback inhibitor of hexokinase.     

Schistosoma mansoni: Ultrastructural damages due to complement on schistosomula in vitro

The ultrastructural damage induced by complement in vitro on the schistosomula of Schistosoma mansoni was studied using transmission and scanning electron microscopy. The sequence of events which leads to the killing of the schistosomula is as follows: (a) the lytic activity starts at the anterior end of the schistosomula; (b) lesions progress simultaneously in two distinct directions: from the anterior to the posterior end, and from the outer membrane to the muscle layer; (c) “bubbles” appear around parasite which might correspond to increased membrane permeability; (d) the lytic activity of the late complement components occur in the syncytium; (e) the schistosomula lose their tegument completely, exposing the muscle layer. These findings and our previous work suggest that the activation of the alternate pathway and late complement components is sufficient, without antibodies or cells, to kill schistosomula in vitro.     

Schistosoma mansoni: Acquired resistance of developing schistosomula to immune attack in vitro

Schistosomula, of Schistosoma mansoni transformed by skin penetration or by mechanical means, have been compared in terms of their susceptibility to in vitro cytotoxic mechanisms, both at 3 hr of age and after culture in the presence or absence of host molecules. Three-hour skin-penetrated schistosomula exhibited a significant level of protection not shown by mechanically transformed individuals. This protection may be correlated with a decreased ability to bind anti-schistosome antibody to their surfaces and to generate C3b molecules as a result of complement activation. Skin worms cultured in the presence of human serum for up to 48 hr showed a significant enhancement of resistance, but slight or no further protection was gained from culture in the absence of host molecules. Mechanically transformed schistosomula cultured for 48 hr in the presence of serum also achieved a significant level of protection but this did not approach that exhibited by the corresponding skin worms; they gained no protection whatsoever from culture in the absence of serum. There are several mechanisms possibly responsible for conferring resistance.     

Schistosoma mansoni: Characterization of antigens in excretions and secretions

Excretory and secretory antigens of Schistosoma mansoni were obtained by in vitro cultivation of worms in Medium H-199, under sterile conditions at 37 C, in the dark, in an atmosphere of 92% air and 8% CO₂. This procedure yielded about 1 μg soluble excretion-secretion products per worm per 24 hr. The composition of the “excretory and secretory antigen” (ESA) preparation is complex. Analysis by isoelectric focusing revealed the presence of about 10 major and about 30 minor protein components. Immunological analysis of the ESA preparation was performed by immunoelectrophoresis. At least five precipitin arcs were seen with infected mouse serum, and seven with rabbit anti-ESA serum. Immunoelectrophoresis of molecular-weight fractions of ESA showed a total of 17 different antigens. One of these antigens was excreted exclusively by female worms. The antibody response in rabbits to preparations obtained by homogenization of adult worms, or by extraction of the tegument, was very different from the response to excretory and secretory antigens. Considerable cross-reactivity between these preparations did, however, occur.     

Schistosoma mansoni: Tegumental surface alterations induced by subcurative doses of the schistosomicide amoscanate

Effects of the administration of a single subcurative dose of a schistosomicidal compound, amoscanate (CGP 4540), on the tegumental surface of adult Schistosoma mansoni were studied using scanning electron microscopy. Worms were recovered from mice between 1 hr and 102 days after treatment. Surface alterations included pronounced swelling, wrinkling and constriction, collapse of sensory bulbs, erosion of large areas of the surface, and attachment of host cells. Different types of lesions of different degrees of intensity were found among worms from the same individual host. Partial and, more rarely, complete repair was noted 62 days after treatment, but even after 102 days not all the lesions had been fully repaired.     

Schistosoma mansoni: Phenol oxidase's role in eggshell formation

Phenol oxidase may be involved in the formation of the eggshell in Schistosoma mansoni. ³H-Labeled female S. mansoni proteins were polymerized in vitro following incubation with S. mansoni phenol oxidase and excess l-tyrosine. Peroxidase inhibitors, autoxidation inhibitors, inhibitors of lipid peroxidation, and inactive analogs of phenol oxidase inhibitors did not inhibit eggshell formation. Fluorescent substances found in eggshell hydrolysates were similar to those formed from the reaction of phenol oxidase-generated quinones with protein-bound lysine. These observations support the classical concept of phenol oxidase-catalyzed protein hardening. However, fluorescent globules of egg material were still formed after the administration of 200 mg/kg of the phenol oxidase inhibitor diethyldithiocarbamate. These globules could not be destroyed by inhibitors of autoxidation and lipid peroxidation.     

Schistosoma mansoni: Cercarial degradation of a radioactively labeled collagen gel

The apparent penetration activity of Schistosoma mansoni cercariae was quantified by means of an in vitro assay with a radioactively labeled Type I collagen gel. Both live cercariae and cercarial preacetabular gland secretions degraded the collagen. The addition of skin lipid or linoleic acid to the gel surface enhanced the degradation by live cercariae.     

Schistosoma mansoni: Infectivity and immunizing effects of in vitro derived schistosomula attenuated by X irradiation

Irradiated and nonirradiated in vitro derived schistosomula of Schistosoma mansoni were injected intraperitoneally into mice. Sixteen percent of nonirradiated schistosomula, 8% of those irradiated with 1000 R, and virtually none of those irradiated with 3000 R and above survived in mice for 5 weeks. However, those irradiated with 3000 R survived in small numbers for shorter periods of time. Schistosomula irradiated with 3000 or 6000 R were used to immunize mice against subsequent infection with cercariae. Prior ip injections of schistosomula irradiated with 3000 R resulted in reductions in worm burden after challenge from 5 to 91%; the observed protection was related to the number of inoculations. The subcutaneous route appeared to be less effective. Schistosomula irradiated with 6000 R produced less protection than those irradiated with 3000 R.