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: The attrition of a challenge infection in mice immunized with highly irradiated live cercariae

Mice immunized percutaneously with 400 Schistosoma mansoni cercariae given 20 kR of ⁶⁰Co irradiation were shown to develop an immunity in which nearly 80% of the parasites that would be expected to survive in control mice were killed. The major attrition of parasites was shown to occur within the first 4 days after challenge. Marked differences in the number of parasites which were recovered from the skin of immune mice and the failure of the majority of parasites to reach the lungs of immune mice indicated that the major site of attrition was in the skin. A further trickle of parasite deaths was evident beyond Day 5, but after Day 14 no further attrition of parasites appeared to occur. Mice immunized in the abdominal skin demonstrated similar levels of immunity whether challenged in the abdominal skin or in the ear. Immunization intramuscularly with irradiated schistosomula induced a much lower level of resistance and the marked parasite attrition in the skin at Day 2 was absent. Immunization with only 50 irradiated cercariae was shown to induce a level of skin immunity equivalent to that seen with 400 irradiated cercariae. The majority of cercariae given 20 kR of ⁶⁰Co irradiation remained in the skin; approximately 2% only reached the lungs. These studies demonstrate that percutaneous immunization of mice with highly irradiated cercariae induced a strong immunity which was largely effective in the skin. This immunity differed from that developed by chronically infected mice where the major attrition of parasites occurs after the lung phase of migration. The results also suggest that the penetration or persistence in the skin of live attenuated schistosomula may play a crucial role in the induction of a high level of skin immunity.     

Schistosoma mansoni: cryopreservation of schistosomules.

Conditions were established for recovery of active schistosomules of Schistosoma mansoni after cryopreservation and storage in liquid nitrogen (?196 C). Schistosomules prepared from cercariae by a shear pressure technique were subjected to a two-step cooling process consisting of a slow cooling rate to an intermediate temperature, followed by rapid quenching of the sample in liquid nitrogen. Overall averages of 39 and 44% of the schistosomules, with a maximum of 88%, were recovered retaining normal activity with cooling rates of 0.4 C/min to ?32 C or 0.8 C/min to ?35 C, respectively. Methanol at 17.5% in Earle's lactalbumin hydrolysate was the freezing medium. As compared with 24 hr storage in liquid nitrogen, no loss in schistosomule motility was observed after 1 month. Following cryopreservation, attenuated schistosomules derived from ⁶⁰Co-irradiated cercariae (50 kR) exhibited structure and activity equivalent to that of unattenuated schistosomules. Infectivity for mice of unattenuated schistosomules derived from ⁶⁰Co-irradiated cercariae (50 Krad) exhibited structure and activity of unfrozen schistosomules ranged from 0.4 to 15.2%.     

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: 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: 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: 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: 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: 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: 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: 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: new method for recording motor activity in vitro

A new method for recording the motility of Schistosoma mansoni in vitro is described. Spontaneous activity of the worm shows contraction similar to peristalsis. The worm responded to electrical stimulation with an immediate contraction that was voltage dependent. Oxamniquine produced an increase in the tonus and spontaneous activity of the worm. This method provides a new experimental model for the study of drugs that interfere with Schistosoma mansoni motility.     

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: 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: schistosomulicidal activity of macrophages isolated from liver granulomas of infected mice

Mice infected with Schistosoma mansoni develop T cell-mediated granulomatous reactions around disseminated parasite eggs. In this study, granuloma-derived leucocytes have been examined for schistosomulicidal capacity by the use of in vitro cytotoxicity assays. Adherent macrophages, that were shown by electron microscopy to exhibit no gross morphological abnormalities, were unable to mediate significant mortality in the absence of serum factors. When cocultured with immune serum and complement, however, these cells killed +/- 26% of the larvae at a cell:target ratio of 5000:1. In contrast, granuloma-derived cell populations that were enriched for eosinophils (50-70% eosinophil content) showed only minimal cytotoxic potential. This may be related to observed structural changes in the eosinophil lysosomal granules, or perhaps to blocking of the cell-surface receptors by immune complexes. It is concluded that granuloma macrophages, activated by egg antigen-sensitised T lymphocytes, may serve as effector cells in immunity to schistosomules.     

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: relationship between parasite age and time of spontaneous elimination from the rat

Laboratory rats infected with Schistosoma mansoni invariably reject the majority of parasites between the fourth and sixth week after infection (self-cure). This investigation was designed to determine whether the timing of rejection is dictated by the rat or by the parasite. Schistosomes were perfused from rats infected 2, 3, or 4 weeks previously and were transferred into the mesenteric veins of normal rats. Recipient animals were perfused at weekly intervals after transfer, and the timing of worm elimination was determined in recipients. It was found that 2-week-old worms were rejected 2 weeks after transfer, 3-week-old worms 1 week after transfer, and 4-week-old worms immediately after transfer. Schistosomes perfused from mice or hamsters and transferred into rats showed the same pattern of worm elimination. It is concluded that at the fourth week of normal schistosome development there is a critical event which makes virtually impossible any further survival of the parasite in laboratory rats.     

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: identification of immunoglobulins associated with the tegument of adult parasites from mice.

Immunocytochemical and immunodiffusion studies were conducted in an attempt to identify the immunoglobulins associated with the tegumental surfaces of Schistosoma mansoni. Peroxidase-labeled monospecific rabbit anti-mouse immunoglobulin class or subclass sera revealed the presence of mouse IgG₁, IgG₂, IgG₃, IgA, and IgM on the surface of adult parasites recovered from mice. These observations were confirmed by double gel diffusion of the various rabbit antisera against an eluate obtained from mouse worms.     

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.