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: Splenic lymphocyte responses of mice after initial exposure to highly irradiated cercariae

Inbred $\text{C}\text{57}\text{B}\text{1}\text{6}$ and CBA mice were immunized with ⁶⁰Co-irradiated (50 kR) Schistosoma mansoni cercariae. Based on the percentage reduction from controls in the numbers of adult parasites developing from a challenge cercarial exposure, the level of protection among immunized $\text{C}\text{57}\text{B}\text{1}\text{6}$ mice ranged from 56 to 74%, and among immunized CBA mice from 10 to 27%. In a longitudinal study, parallel in vitro comparisons of mitogen- and antigen-stimulated lymphocyte proliferative responses were performed with spleen cells from immunized and control mice of both strains. In contrast to decreased mitogen reactivity during a chronic, patent infection, immunization with irradiated cercariae resulted in no alteration in PHA and LPS responses in the reactivity of either strain. A vigorous antigen-specific reactivity was noted in the responses of immunized CBA mice. Additionally, a biphasic pattern of responsiveness characterized the CBA responses to antigens of cercarial, adult worm, or schistosomal egg origin. In comparison, there was a greatly diminished reactivity in immunized $\text{C}\text{57}\text{B}\text{1}\text{6}$ mice to the same antigens. Therefore, no obvious correlation existed in this model between the relative magnitude of antigen-specific responses between the two strains and the level of anti-schistosome immunity induced.     

Schistosoma mansoni: vaccination of mice with cryopreserved irradiated schistosomules.

In our earlier experiments, NIH/Nmri (CV) mice developed protective immunity to a Schistosoma mansoni cercarial challenge when previously exposed percutaneously to highly ⁶⁰Co-irradiated homologous cercariae. Experiments reported here were conducted to assess the immunogenicity of unfrozen and frozen and thawed schistosomules derived from ⁶⁰Co-irradiated cercariae (irradiated schistosomules). Immunization of NIH/Nmri (CV) mice by ⁶⁰Co-irradiated unfrozen schistosomules reduced worm burdens from a subsequent percutaneous challenge with normal cercariae by 41 to 72%. Immunogenicity was not narrowly dependent on irradiation dose rates between 1 and 8 kR/min, or on the total dose of irradiation given the schistosomules between 25 and 50 kR. Comparable protective immunity developed after injection of irradiated schistosomules which had been frozen to ?196 C in liquid nitrogen and thawed. Cryopreservation appears to offer a solution to the problem of storage of attenuated, immunogenic S. mansoni schistosomules.     

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.     

Schistosoma mansoni: Role in vivo of complement in primary infection of mice

The role of complement in the control of the primary Schistosoma mansoni infection in mice was investigated in vivo. The number of recovered adult schistosomes 6–7 weeks postinfection was used as a parasitological criterion of immunity. No significant difference in the worm burden was observed between C5-sufficient and C5-deficient mice. In contrast, when cobra venom factor (CVF) was injected into normal or C5-deficient mice 24 hr before challenge, a significant increase of the worm burden was noticed in comparison to the untreated mice. These results indicated that, although C5 and probably the late complement components are not essential for the control of the primary infection, the alternative pathway and some of its components are involved. In fact, the injection of C3 2 hr before infection of CVF-treated mice completely restored the immunity. A role for C3, in association with effector cells, in the nonspecific immunity occurring in the first hours after a primary S. mansoni infection is suggested.     

Schistosoma mansoni: Resistance to an infection with cercariae induced by the transfer of live adult worms to the rat

Partial resistance to an infection by cercariae of S. mansoni was induced in Sprague-Dawley rats by the surgical transfer into a mesenteric vein of live adult worms, recovered from infected mice by portal perfusion. A biological index, correlating with resistance, was sought in order to be used as a guide for developing an immunization protocol. Concurrent induction of peripheral eosinophilia and anti-worm antibodies in recipients of live worms correlated with induction of resistance to a subsequent cercarial infection. This response characteristic may provide a useful, preliminary index for screening worm antigen preparations intended for use in protective immunization protocols.     

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: Post-transformational surface changes in schistosomula grown in vitro and in mice

The development of schistosomula during the first 4 days after transformation from cercariae has been examined in parasites isolated from the lungs of mice and in organisms cultured in lactalbumin and rabbit serum or in the defined serum-free medium, RPMI 1640. The development of organisms grown under all three conditions was the same. Schistosomula increased in length from 67 to 110 μm and decreased in width from 24 to 18 μm, so that the volume remained constant at approximately 2.7 × 10⁴ μm³. The increase in length occurred mainly in the torso or posterior three-quarters of the worm which increased from 49 to 88 μm or 80%, whereas the head increased from 18 to 22 μm or 22%. The spines were lost from the surface that was most rapidly lengthening by gradual resorption into the tegument and were replaced by pits mainly during the first 3 days. These changes resulted in a 325% increase in the surface area of the schistosomula, from 1.2 × 10⁴ to 3.9 × 10⁴ μm². In addition, the openings of the acetabular ducts, the ventral sucker, and the tail socket all became smaller and flatter over the four-day period. Internally, the major changes were the loss of the acetabular ducts in the pre- and post-acetabular glands and an increase in size of the caecum. In summary, these experiments show that the surface of the schistosomulum is extensively remodeled before intravascular migration occurs and demonstrate the efficacy of RPMI 1640 as a culture medium for schistosomula in the first 4 days after transformation.     

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: Anodic polysaccharide antigen in glomerular immune deposits of mice with unisexual infections

In mice infected with unisexual Schistosoma mansoni, circulating anodic antigen was detected by immunofluorescence in glomeruli of 20 out of 22 animals from 7 to 30 weeks after infection. Circulating anodic antigen was present as finely granular deposits in the mesangium. The amount of circulating anodic antigen in the glomeruli was not clearly related to the worm burden but it increased during the course of the infection. These circulating anodic antigen deposits were accompanied by deposits of immunoglobulins, sometimes found in the same precise localization, and of complement. They probably represent the antigen part of immune complexes. Circulating anodic antigen appears to be a major candidate among the antigens involved in schistosomal glomerulopathy.     

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: 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 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: Ultrastructure of adults from mice treated with oxamniquine

The in vivo effects of a single oral dose (50 mg/kg) of oxamniquine on the ultrastructure of Schistosoma mansoni were investigated. In male worms, severe disruption of the tegument and gastrodermis took place, and extensive extracellular spaces developed between the cells of the internal tissues. Elimination of the damaged worms was associated with complete tegumental breakdown and encapsulation by host cells. A small proportion of females showed similar drug-induced changes and were also eliminated. In the residual females, no drug-induced morphological damage was observed even after a second dose of oxamniquine. However, these females became much reduced in size, and regression of the organs of the reproductive system took place. It is suggested that such regressive changes resulted from discontinued male stimulation rather than the direct effect of oxamniquine.     

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: 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: Complement and antibody damage,mediated by human eosinophils and neutrophils,in killing schistosomula in vitro

The time and course of damage to Schistosoma mansoni schistosomula mediated by human eosinophils and neutrophils and by antibody (A) and/or complement (C) was studied. The rate of schistosomula death was significantly higher in the complement containing systems (i.e., “A + C” or “C alone”) when compared to A alone. In general, at all the time points studied, the percentage of killing in the three systems was A + C > C alone > A alone irrespective of whether the effector cells were neutrophils or eosinophils. Preferential killing of schistosomula by eosinophils, as compared to neutrophils, was observed with C alone and A + C, but only when eosinophils and neutrophils from the same donor were compared. In contrast, eosinophils and neutrophils appeared to be equally effective in killing antibody-coated schistosomula.A comparison was made of the susceptibility to killing of schistosomula prepared mechanically or by skin penetration. There was no appreciable difference in terms of susceptibility to killing by the various combinations of eosinophils, neutrophils, antibody, and complement between these two forms of schistosomula.The preferential killing of complement-coated, as compared to antibody-coated schistosomula by eosinophils appears to be relatively rapid, an observation which may be of significance both in natural and acquired immunity to migrating larval helminths.     

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: 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: Adult worm chemoattraction,with and without barriers

Heterosexual and homosexual chemoattraction studies were done with 8- to 12-week-old Schistosoma mansoni adults in linear chambers containing Earle's saline, in the absence and presence of both perforated and unperforated dialysis sac chimney barriers. In the absence of barriers attraction was seen in all heterosexual and homosexual combinations. After 90 min of observation at 37 C, organisms maintained at 4 or 37 C for at least 1 hr prior to the assay showed heterosexual attraction similar to those used immediately after perfusion. There was significantly more heterosexual attraction between worms from same than different pairs. In the presence of perforated barriers the greatest attraction was heterosexual, with males moving toward females; heterosexual attraction with females moving toward males was approximately equal to homosexual female attraction, and male homosexual attraction was not seen. In the presence of unperforated barriers the greatest attraction of females toward males occurred during the first 0.5 hr; within 3 to 4 hr females were no longer significantly attracted to males. However, after 0.5 hr males were more significantly attracted to females in unperforated than perforated chimneys. These studies demonstrate that adult schistosomes attract each other in vitro, and that there is a chemical basis for this attraction.