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: vaccination of mice with 10-krad-irradiated, cryopreserved schistosomules

Protection against a Schistosoma mansoni cercarial challenge was evaluated in mice immunized with a vaccine composed of 10-krad-irradiated, cryopreserved schistosomules. The level of resistance induced in C57B1/6 or NMRI (CV) mice increased with the number of schistosomules injected. Up to 83% reduction in challenge worm burden was achieved when 5000 schistosomules were injected per mouse. Intramuscular injection of the vaccine was superior to subcutaneous. Multiple immunizations, up to 3 at 4-week intervals, did not increase the resistance induced by a single immunization. A high level of protection developed in as little as 2 weeks and was maintained through at least 12 weeks postimmunization. The vaccine irradiated with 10 krad from either a 60-cobalt or 137-cesium source induced equivalent levels of resistance, and no differences were found in the immunogenicity of vaccines comprised of organisms irradiated as cercariae or as 1- to 3-hr-old schistosomules. These findings are basic to the development of a cryopreserved, live vaccine against schistosomiasis of humans or domestic animals.     

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: stimulus and transformation of cercariae into schistosomules

Schistosoma mansoni schistosomules prepared from cercariae by seven in vitro techniques had not all reached the same state of development at the end of the incubation period as scored by seven parameters: water tolerance; Cercarienhüllen Reaktion; presence of the glycocalyx; condition of the surface membrane; nuclear state; granule migration; and cryopreservability. At the end of the specific incubation period for each technique, the level of development was judged with respect to schistosomules which had developed in situ for 1 hr after penetration of the ear skin of mice. In descending order of their correspondence to in vivo schistosomules, those derived in vitro (by the procedures listed) ranked as follows: first, penetration of dried rat skin; second, centrifuging and vortexing, or incubation in serum-supplemented medium; and third, syringe passage, omnimixing, centrifuging, and incubating, or incubating alone. The only treatment common to all techniques was incubation in 37 C culture medium for 2 hr or more. This is suggested as the stimulus for the cercaria-to-schistosomule transformation.     

Schistosoma mansoni: uptake of exogenous hemeproteins by schistosomules grown in vitro

The uptake and fate of the hemeproteins horseradish peroxidase (HRPO) and hemoglobin (Hb) by schistosomules of Schistosoma mansoni maintained in vitro were studied by electron microscopy and cytochemical techniques. After administration of HRPO, reaction product was observed initially in the lumen of the digestive tract, and, after 2 hr of feeding, reaction product was also visible in the cytoplasm of the gastrodermis. There was no evidence of pinocytosis. After administration of Hb, reaction product was observed only in the lumen of the digestive tract. As is found following red blood cell feeding, digestive pigment was formed in the lumen of the gut following Hb feeding. The possible significance of these findings is discussed.     

Schistosoma mansoni: Interactive effects of irradiation and cryopreservation on parasite maturation and immunization of mice

Mechanically transformed schistosomula of Schistosoma mansoni were irradiated with levels of ⁶⁰Co irradiation between 2.5 and 54 krad, cryopreserved by the two-step addition of ethanediol and rapid cooling technique, and were injected intramuscularly into groups of mice which were perfused 40 days later. The schistosomula were either irradiated and then cryopreserved (IC) or cryopreserved and then irradiated in the frozen state (CI). Development into adult worms was prevented with 4 krad for IC schistosomula, but for CI schistosomula a small number of worms (1.6%) was recovered using 8.8 krad. A dose of 4 krad was sufficient to prevent development of unfrozen controls (I), but for schistosomula irradiated while exposed to ethanediol (EI), a dose of 7 krad was required. Using the different protocols, the peak levels of protection against a challenge infection were achieved with 9 (IC) and 16 krad (CI), compared to 20 krad for unfrozen schistosomula (I) reported previously. The highest level of protection (65%) was achieved with CI schistosomula. Possible interactions between the radioprotective and damaging effects of cryopreservation are discussed.     

Schistosoma mansoni: patter of release of secretory products.

Adult Schistosoma mansoni were maintained in vitro for 1 hr with radioactively labeled precursors of protein, glycoprotein, and polysaccharides. The worms were then washed extensively and the supernates analyzed. The precursors N-acetylglucosamine, N-acetylgalactosamine, glucosamine, galactosamine, glucose, leucine, and fucose were incorporated into the worms and both large and small molecular weight products accumulated in the supernatant. For all the precursors except fucose, there was an initial rapid and then slower phase of release for both the large and small molecular weight materials. The amount of label retained by the worms as well as the proportion excreted as large molecular weight material was characteristic for the precursor used. In contrast, the products of fucose were released within 4 to 6 hr and therefore only exhibited the early secretory phase. There was no retention of fucose by the worms. Hydrolysis of large molecular weight products revealed that the N-acetylglucosamine-derived material was incorporated as amino sugars and fucose was incorporated as fucose. Therefore, N-acetylglucosamine and fucose precursors can specifically label secretory glycoproteins of schistosomes in a manner similar to that in mammalian systems.     

Schistosoma mansoni: mice infected with different worm strains.

Infections with five geographical strains and substrains of Schistosoma mansoni were compared in mice. Two substrains (Lc-1 and Lt-1) were derived from the parent (L-1) St. Lucian strain on the basis of differing infectivity for various snail strains. The Puerto Rican strains (PR-1 and PR-2) were obtained with an interval of 25 years. Consistent differences among the lines were found in egg distribution and numbers of eggs in tissues and feces. One Puerto Rican strain (PR-2) and one St. Lucian substrain (Lc-1) had longer prepatent periods than the other strains. Mice infected with the PR-1 strain consistently had the highest egg accumulation in the tissues per worm pair. Relatively few eggs were passed in the feces of the Lt-1 strain. By Week 9 of infection, eggs were noted in the spleens of mice carrying each of the strains and substrains.     

Schistosoma mansoni: peripheral and tissue eosinophilia in infected rats.

Peripheral eosinophilia is induced in Sprague-Dawley rats following infection with cercariae of Schistosoma mansoni. Beginning 3 weeks after infection, peripheral eosinophil levels rise above the baseline range; they reach peak values during the fifth week. Following a decline from peak values, peripheral eosinophil levels remain elevated and are observed to fluctuate for the next 5 months. The magnitudes of both the initial peak response at 5 weeks and the subsequent chronic level of peripheral eosinophils are dependent upon dose of cercariae. The initial peak response phase of peripheral eosinophilia coincides in time with the period of adult worm elimination (Weeks 4–6) in the schistosome-infected rat. Histological examination of the liver at 5 weeks after infection reveals eosinophil-rich inflammatory reactions associated with both live and dead worms residing in the portal blood vessels. Around live worms the inflammatory cells are localized in a perivascular arrangement; around dead worms these cells are in the vascular lumen in contact with destroyed worms. The chronic phase of peripheral eosinophilia is associated, in part, with inflammatory reactions surrounding eggs deposited in the liver by the few worm pairs which survive more than 6 weeks and remain within the liver. Histological examination during this period reveals granulomatous lesions within the liver surrounding eggs and dead worms. The granulomas are predominately monocytic (lymphocytes, macrophages) at 11 and 16 weeks. The initial peak response phase of peripheral eosinophilia appears to be a marker for tissue-localized reactions of eosinophils with worms. There are relationships between inflammatory reactions and survival of adult worms.     

Schistosoma mansoni: circulating antigens and immune complexes in infected mice.

Circulating schistosome antigens (CSA) and circulating immune complexes (CIC) were investigated during the course of Schistosoma mansoni infection in mice. The radioimmunoprecipitation-polyethylene glycol (PEG) assay (RIPEGA) with [¹²⁵I]anti-S. mansoni antibodies or [¹²⁵I] anti-antigen “4” antibodies detected, respectively, total CSA and antigen “4” in serum and in 3% polyethylene glycol-precipitated CIC from infected mice. Complement fixation test and [¹²⁵I] C_1q-binding test revealed, respectively, an anticomplementary activity and the presence of C_1q-binding CIC. All these substances appeared in infected mice at approximately the same period, i.e., between the 40th- and the 55th- day postinfection. No correlation was observed between the detection of anticomplementary active substances and C_1q-binding CIC. In contrast, a close relationship was noticed between CSA and complement-activating material during the course of the infection. This suggests that substances with anticomplementary activity in serum from infected mice could be one or various CSA. A close correlation was also observed between C_1q-binding CIC and free or “complexed” antigen “4.” This observation supports well the possibility that antigen “4” is one of the major complexed circulating antigen present in schistosomiasis. The immunoglobulins G₁, G_2a, M, and A were also characterized in 3% PEG-precipitated CIC from infected mice during the period in which we detected C_1q-binding CIC. The roles played by specific S. mansoni CIC in either schistosomal nephropathy or protective mechanisms to a challenge infection in mice are discussed.     

Schistosoma mansoni: complement and cercarial tail loss during in vitro transformation.

When Schistosoma mansoni cercariae are incubated at 37 C in media containing serum, the organisms lose their tails and change into viable, infective schistosomula. Tail loss does not occur in the absence of serum, or when the serum is heat inactivated. In the present studies, tail loss during in vitro conversion was shown to be complement dependent. The capacity of fresh serum to promote tail loss was markedly suppressed or abolished by cobra venom factor, zymosan, Sepharose CL-4B AND anti-C3 antibody. The alternative rather than the classic complement pathway appeared to be responsible since (1) binding of anti-C3 to cercariae required magnesium, but not calcium; (2) both C4-deficient serum and C2-deficient serum supported tail loss; but (3) human serum heated to 50 C for 20 min to inactivate Factor B did not support tail loss. Cercarial tail loss also required the terminal complement components C5 through C8. The extent and rate of tail loss was normal in agammaglobulinemic sera indicating that the complement effect was not antibody dependent.     

Schistosoma mansoni: effects of antimony on immature and adult worms.

Immature Schistosoma mansoni in mice are less susceptible to antimony therapy than adult worms. KSb tartrate inhibited phosphofructokinase (PFK) (EC 2.7.1.11) to a greater extent in extracts of 3-week-old worms than adults, and inhibited production of lactate in both immature and adult worms in vitro. In vivo, KSb tartrate was accumulated similarly by 3-week-old worms and by adults: measurements of hexosephosphate following drug treatment suggested similar inhibition of PFK in the two worm stages. If antimony acts by inhibition of PFK it is not clear why the young worms are more resistant to chemotherapy than adults.     

Schistosoma mansoni: species-selective response to N-chloroethyl-acetylcholine derivatives

Three new acetylcholine mustard analogs were tested on schistosome and vertebrate neuromuscular preparations. These compounds extend a previously reported structure-activity series. The new compounds investigated include isopropyl-, cyclohexyl-, and benzyl-2-acetoxyethyl-2'-chloroethylamine (PrM, ChM, and BzM). In schistosome motor activity studies, all of the compounds caused irreversible reductions in the activity of Schistosoma mansoni after 1 hr exposure followed by 19 hr in drug-free medium. Under nonlethal conditions of dosage and exposure time, all compounds blocked carbachol-induced paralysis, indicating a possible action at schistosome cholinergic sites. All the compounds also reduced the labeling of schistosomes by dimethylaminonapthalene-5-sulfonamidoethyl dimethylamine hydrochloride (DDNS), a fluorescent ACh analog. In isolated vertebrate tissue experiments. PrM and ChM had slight agonist activity in the guinea pig ileum, and only PrM had agonist activity in the frog rectus abdominis preparation. PrM and BzM were found to antagonize ACh responses in the guinea pig ileum. Exposure of vertebrate tissues for 1 hr to high concentrations of ChM caused no long-lasting inhibition of ACh, pilocarpine, and serotonin (5HT) responses. Histamine responses were slightly reduced from control. Following 1 hr exposure of vertebrate tissues to PrM, initial reductions in all responses were seen, followed by recoveries to near control. BzM treatment, however, reduced all responses far below control; the tissues did not recover even after washing for 2 hr. It is concluded that ChM and, to a lesser extent, PrM, had a greater effect on schistosomes than on vertebrate tissues. BzM did not display species selectivity in this favorable direction.     

Schistosoma mansoni: neurotransmitters, longitudinal musculature and effects of electrical stimulation

Longitudinal muscle shortening of adult male Schistosoma mansoni is produced by electrical stimulation. Responses are frequency and strength dependent. Neither 5-hydroxytryptamine (5-HT) antagonists nor dopamine interfere with the response. 5-HT does not enhance it. Carbachol (10^?4M), eserine (10^?6M), and metrifonate (10^?5M) block the response but acetylcholine alone has no affect. Atropine (10^?4M) partially counteracts the effects of carbachol. Hyperosmotic sucrose but not urea blocks the stimulation response. It is concluded that the response is nonneuronally conducted via a pathway involving gap junctions and that neurotransmitters probably act as modulators of motor activity rather than as initiators of it.     

Schistosoma mansoni: partial purification and properties of ornithine-delta-transaminase.

Ornithine-δ-transaminase (OTA) (EC 2.6.1.13) was isolated from Schistosoma mansoni and purified more than 16-fold. Treatment of the worm homogenate with 0.4% deoxycholate (DOC) in the presence of 0.8 M KC1 and 0.15 M NaCl at pH 8.3 resulted in solubilization of 85% of the enzyme. Sonication and high-speed centrifugation were unnecessary. The solubilization procedure and the subsequent purification steps required the presence of the coenzyme pyridoxal phosphate. The optimal pH for OTA was 8.5 and the optimal incubation temperature was 55 C. Michaelis-Menten constants (K_m) for ornithine and α-ketoglutarate were 1.53 mM and 2.07 mM, respectively, in enzyme preparations with a specific activity of 22–29 μmoles/hr/mg protein. The enzyme showed a high affinity for α-ketoglutarate but considerably less affinity for oxaloacetate and pyruvate. High concentrations of α-ketoglutarate and ornithine inhibited the OTA activity. Similarly inhibitory were the structurally related amino acids isoleucine and serine and also oxaloacetate. The K_m for α-ketoglutarate in the presence of oxaloacetate was 1.3 mM and the V_max was 8.38 μmoles/hr/mg protein.     

Schistosoma mansoni: physical and chemical factors affecting the mechanical properties of the adult male musculature.

The response of the musculature of male Schistosoma mansoni to various physical and ionic environments was determined. Contractile activity and tension of the parasite's musculature decreased when they were incubated in buffered salt solutions having an osmolality greater than 300 mOsm, a pH less than 6.8, or a temperature greater than 39 C. Except for potassium, high concentrations of inorganic ions reduced the tension of the parasite's musculature; high concentrations of potassium increased tension. In general the contraction rate of the male schistosome decreased when the concentration of an inorganic ion was below or above that found in Hanks' balanced salts solution. These results indicate that the musculature of S. mansoni is similar to smooth muscle found in mammals.     

Schistosoma mansoni: immunization of cynomolgus monkeys by injection of irradiated schistosomula.

Although the immunization of primates with irradiated schistosome cercariae has been demonstrated, no success has been reported by injection with the irradiated schistosomule stage. The present investigation was designed to test whether cynomolgus monkeys could be protectively immunized with ⁶⁰Co-irradiated Schistosoma mansoni schistosomula. Monkeys injected once with 10⁴ irradiated schistosomula (50 krad at 4 krad/min) had 52% fewer challenge worms than the control group at necropsy. Four immunizations did not induce a higher level of resistance. At 50 days post-challenge, the immunized monkeys excreted 80% fewer eggs than did the control animals. An attempt to enhance irradiated schistosomule-induced protection with tetramisole · HCl was unsuccessful.     

Schistosoma mansoni: description of the head gland of cercariae and schistosomules at the ultrastructural level.

The head gland of the cercaria of Schistosoma mansoni appears to be a relatively large unicellular entity consisting of a fundus tapering into a system of multiple ducts that opens into the integument at the anterior end of the oral sucker. The fundus is located in the posteriodorsal area of the oral sucker and contains most of the secretory granules. The ducts are usually narrow and devoid of secretory granules especially near their integumental junctions in the cercaria. In Schistosomules of S. mansoni the fundus is reduced and the ducts are distended as secretory granules move en masse into the integument during the penetration of cercaria into their host where they may provide material for repair of the integument of the oral sucker damaged during penetration. The head gland has a strong affinity for luxol fast blue and acid hematin stains which suggests the presence of phospholipids.