Schistosoma mansoni and Fasciola hepatica: Cross-resistance in mice

Cross-resistance in Schistosoma mansoni and Fasciola hepatica infections were studied in mice. A primary infection of S. mansoni, 7 to 28 days old, did not stimulate a significant level of resistance to heterologous challenge with F. hepatica. In contrast, in older S. mansoni infections (54–65 days old) there was a significant level of resistance to a challenge with F. hepatica. The F. hepatica worm burden was reduced by 34.0 to 72.5% in separate experiments. Challenge infection with F. hepatica did not influence the number of S. mansoni in primary infections. No heterologous resistance to S. mansoni was found in mice with 7- and 23-day-old F. hepatica infections. However, primary infections with F. hepatica, 28, 32, 42, and 50 days old, conferred significant resistance to a heterologous challenge with S. mansoni. The established schistosome worm burden was reduced by 41.5 to 50.4%. In no case was the primary F. hepatica burden reciprocally influenced by challenge infection with S. mansoni.     

Schistosoma mansoni and Schistosoma haematobium: Differences in development

Growth and maturation of the Puerto Rico strain of Schistosoma mansoni in mice and the Ghana strain of Schistosoma haematobium in hamsters were compared beginning 19 days after infection. In S. mansoni, optimum development was determined, with copulation first observed on Day 25, egg shell protein formation observed on Day 28, and oviposition occurring on Day 30. In infections of S. haematobium, copulation first occurred on Day 29. Egg shell proteins were first formed on Day 45, and egg production occurred on Day 60. Examination of unisexual and bisexual infections showed that maturation of the vitellaria can be more easily assessed by autofluorescence of the protein globules than by the traditional diazonium salt stains. Autofluorescence of living worms with mature vitellaria allows subsequent examination by electron microscopy, and therefore permits evaluation at a subcellular level.     

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.     

Fasciola hepatica: Carbohydrate metabolism of the adult

Glycogen (or exogenous glucose) was the only energy source utilized by adult Fasciola hepatica under a number of different incubation conditions. When exogenous glucose was present in the incubation medium, significant amounts of lactate were excreted. Anaerobically, in the presence of glucose, lactate accounted for 20% of the total end products measured. In the absence of glucose, organic acid production accounted for approximately 60% of glycogen carbon utilized; this value was reduced to 40% in the presence of exogenous glucose. There was no appreciable Pasteur effect.     

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.     

Fasciola hepatica: Development of caecal epithelium during migration in the mouse

Juveniles of Fasciola hepatica excysted and penetrated the intestinal wall of mice within 2 hr of infection. Approximately 70% of the administered dose was present in the abdominal cavity after 7 hr. Many large (1.3 μm in diam) secretory granules, stored in the paired caeca of the newly excysted juvenile were used up during the penetration of the intestinal wall. The caecal cells of juveniles newly arrived in the abdominal cavity 2 and 12 hr p.i. (postinfection) did not have any of the usual morphological features associated with an absorptive function and the caeca may therefore be regarded as having a role in penetration. Parasites recovered from the abdominal cavity 1 day p.i. had caecal cells which for the first time exhibited morphological features of an absorptive function; an apical plasma membrane amplified into occasional short lamellae and junctional complexes with parenchymal cells at the basal plasma membrane. These cells carried out synthesis and secretion of the large, 1.3 μm, granules throughout the parasite's migration to the liver capsule. These large granules were absent from the caecal cells of juveniles which had become established in the liver capsule. Also on arrival in the liver capsule the caecal cells transformed into an adult-like morphology in that their granular endoplasmic reticulum gained a basal-apical orientation and their apical plasma membranes were amplified into tall regular lamellae. The transformed cells synthesized and secreted small (0.5 μm in diam) granules similar in size to those produced by adult cells. Growth of the caeca occurred by mitotic division of undifferentiated cells observed in the epithelium and subsequent redifferentiation of daughter cells. Possible origins of the undifferentiated cells are discussed.     

Fasciola hepatica: Energy sources and metabolism

Gas chromatographic determination of biliary glucose in the rat showed only low levels were present. Although flukes took up glucose from the bile in vivo, biliary glucose could not be a major energy source as occurs in Hédon Fleig solution in vitro. Alanine, arginine, glutamate, histidine, phenylalanine, and serine were not metabolised in vitro to volatile fatty acids, and mixtures of peptides or amino acids failed to spare glycogen breakdown in the absence of glucose. Although glycerol was metabolised in vitro, the main energy source of the fluke in vivo has not been identified. Labelling studies confirmed that glucose taken up in vitro is degraded via glycolysis rather than the pentose phosphate pathway.     

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.     

Fasciola hepatica: The effect of challenge by the subcutaneous or intraperitoneal route with living adult flukes in sensitized rats

When rats, sensitized either by subcutaneous implantation of adult F. hepatica or by a normal oral infection with F. hepatica metacercariae, were challenged by implanting adult flukes in the peritoneal cavity, 23% of these flukes were killed in rats sensitized by subcutaneous implantation and 71% in the rats sensitized by the oral route. In contrast neither of these sensitization routes were effective against subcutaneous challenge with adult fluke. Histological evidence suggested that about half of the dead flukes found were killed shortly after transfer and these flukes were surrounded with mononuclear cells. The remaining dead flukes appear to have died after becoming surrounded with a cyst. These latter flukes were surrounded by neutrophils and this cell type was very prominent in the cysts of sensitized rats.     

Fasciola hepatica: A proteolytic digestive enzyme

A proteolytic enzyme in the gut exudate of the common liver fluke has been purified. The enzyme is specific for globin with a pH optimum of 3.9–4.0 and breaks the protein down to peptides and a small percentage of free amino acids. Collagenase activity at pH 7.5 copurifies with the main globinolytic enzyme. The enzyme has a molecular weight of 12,000 and does not appear to be antigenic in fluke-infected animals.     

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.     

Fasciola hepatica: Stereological analysis of vitelline cell development

Cytomorphosis of vitelline cells in Fasciola hepatica has been studied quantitatively by means of a Kontron Videoplan computerized image analyser. The process of vitelline cell development was subdivided into four characteristic phases: the stem cell, the intermediate type 1 cell, the intermediate type 2 cell, and the mature cell. The whole cell and the following constituent organelles, the nucleus, nucleolus, mitochondria, granular endoplasmic reticulum (GER), secretory granules, glycogen, heterophagosomes, and lipid, were analysed at each phase. Results indicated that there was a significant increase in nuclear size between the two intermediate cell stages, and a significant increase in nucleolar size between the stem cell and intermediate type 1 cell; the changes were related to possible gene activation, ribosome production, and cell synthesis required for both cell growth and secretory production. Mitochondria increased in number and showed changes in volume and surface area of whole organelles and their cristae such that these correlated with the energy demands of growth and synthesis. The GER increased its average total volume some 16 times and its average total surface area some 25 times between the stem cell and mature cell stages. Even this increase masked the true extent of membrane production by this system, since membrane was transferred to secretory granules; in reality the GER or GER-derived membranes increased more than 42 times between the stem cell and mature cell stages. Shell globules and glycogen eventually contributed 21 and 20%, respectively, to cytoplasmic volume, while heterophagosomes contributed 14% and lipid only 3%. Residual cytoplasm was shown to be an important component in cytomorphosis, comprising never less than 40% of the total cytoplasmic volume and it was shown to increase prior to or parallel with the development and expansion of other systems.     

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: Tegumental damage as a consequence of lectin binding

Adult worms of Schistosoma mansoni exhibit gross tegumental damage following incubation in concanavalin A or Ricinus communis agglutinin. However, incubation with wheat germ agglutinin induces only minimal surface damage, while soybean agglutinin has no damaging effect upon the worms. Damage induced by Ricinus communis agglutinin or concanavalin A may be prevented by the addition of the appropriate competing sugar. In contrast, incubation of 3-hr artificially transformed schistosomula in concanavalin A and other lectins does not produce any disruption of the tegument. These results indicate that the surface membrane of the adult schistosome is readily disrupted by ligand binding and appears to be particularly sensitive and fragile. The membrane of the schistosomulum, however, is more resistant to the effects of lectin binding. Adult worms incubated in culture medium alone (ELAC or RPMI 1640) show background changes which seem to be related to the tonicity of the medium. Such results advocate that preliminary assessment of schistosome integrity be carried out prior to any experimental procedures which preclude the addition of serum to the basic incubation medium. Schistosomula do not exhibit comparable sensitivity.     

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 and S. haematobium: Calcium metabolism of the vitelline cell

The mature vitelline cell in Schistosoma mansoni and S. haematobium contains inclusions identified as calcareous corpuscles. They originate in the dilated cisternae of the granular endoplasmic reticulum and are eventually released when the vitelline cell starts to disintegrate in the fully formed egg capsule. X-ray microprobe analysis of single-fixed but conventionally prepared specimens, as well as of material quenched in liquid nitrogen slush and cryosectioned, indicated that the corpuscles contained phosphorus, calcium, and magnesium. The debris surrounding the developing embryo within the egg capsule also contained the elements phosphorus and calcium. Dosage of infected mice with Astiban produced an increase in total calcium content.     

Trypanosoma musculi and Trichinella spirails: Concomitant infections and selection for resistance genotypes in mice

Trypanosoma musculi infections were given to mice of different strains before, at the same time, and after an infection with 400 Trichinella spiralis. Examined parameters of the host response to T. spiralis were worm rejection, antifecundity responses, development of immunological memory, and muscle larvae burden. After dual infection, each mouse strain showed characteristic effects on resistance to T. spiralis. This was due to a dynamic interaction between the genes controlling rejection of T. spiralis and those influencing T. musculi growth. C3H mice develop high trypanosome parasitemias. This impairs worm expulsion and the development of memory to T. spiralis when Trypanosoma infections take place on the same day or 7 days before. The C57B1/6 mouse develops low parasitemias and T. musculi infections on the same day, or 7 days before T. spiralis, delaying worm rejection only slightly despite the overall weak capacity of B6 mice to expel worms. NFR-strain mice are strong responders to T. spiralis and also develop low parasitemias. Trypanosome infections on the same day, or after T. spiralis, produce a delay in worm rejection; the former is comparable to C3H mice. However, NFR mice alone showed enhanced rejection of worm when T. musculi infections preceded T. spiralis by 7 days. An unusual feature of C3H mice was that T. musculi infections 7 days before T. spiralis increased antifecundity responses at the same time that worm expulsion was inhibited. Trypanosome infections can therefore modulate distinct antihelminth immune responses in different directions simultaneously. The different outcomes of dual infections compared with single infections provides another selective mechanism by which genetic polymorphisms can be established and maintained in the vertebrate host.     

Enzyme levels in homogenates of liver from mice infected with Schistosoma mansoni and from uninfected mice

Enzymatic and histopathological study of the livers of 300 white mice infected with Egyptian strain of Schistosoma mansoni and of 100 noninfected control animals was carried out for a follow up period of 6 months.Significant diminution of pseudocholine esterase activity was noticed as early as the second week after infection, denoting defective synthetic function of the liver. Significant decrease of transaminases was also noticed during the fifth week, and showed correlation with the histopathological findings of cell necrosis. Glutamic oxalacetic transaminase appears to be a better index of liver cell integrity than glutamic pyruvic transaminase.Lactic dehydrogenase was only moderately diminished during the third and fourth months.Alkaline phosphatase was strikingly increased in hepatic schistosomiasis and occurred as early as the second week. Increased enzyme production and release in the circulation has been suggested.These results confirm the affection of the liver cell during infection with Schistosoma mansoni.     

Schistosoma mansoni Annexin 2: Molecular characterization and immunolocalization

We here describe the cloning and characterization of the Schistosoma mansoni Annexin 2, previously identified in the tegument by proteomic studies, and as an up-regulated gene in schistosomulum stage by microarray data. In silico analysis predicts a conserved core containing four repeat domains of Annexin (ANX) and a variable N-terminal region similar to that described for mammalian isoforms. Real-time RT-PCR and Western blot analysis determined that S. mansoni Annexin 2 is significantly up-regulated in the transition from free-living cercaria to schistosomulum and adult worm parasitic stages. Immunolocalization experiments and tegument membrane preparations confirmed Annexin 2 as a protein mainly localized in the tegument of schistosomula and adult worms. Furthermore, it binds to the tegument surface membranes in a calcium-dependent manner. These results suggest that S. mansoni Annexin 2 is closely associated to the tegument arrangement, being a potential target for immune intervention.     

Fasciola hepatica: Migration of newly excysted juveniles in resistant rats

Using transmission and scanning electron microscopy, the early migration of juvenile Fasciola hepatica was examined in naive and resistant rats. In naive rats, the migration of flukes to the peritoneal cavity was uneventful. In resistant rats, flukes were rapidly coated with antibody whilst still in the gut lumen and a proportion of the flukes were unable to penetrate the intestinal wall. Those that did penetrate were unharmed as they crossed the gut wall, but on entering the peritoneal cavity they were coated with antibody and host cells including eosinophils, neutrophils, macrophages, and mast cells. Eosinophils were seen degranulating onto the fluke surface, and this appeared to result in the erosion of the tegumental syncytium.