Acquisition of host proteins by the tegument of Schistosoma mansoni recovered from rats

Four-week-old Schistosoma mansoni collected from experimentally infected albino rats were shown by a fluorescent antibody technique to possess several serum proteins of host origin, including IgM, IgG, complement and red blood cell components. The capacity of these rat-derived worms to acquire and shed host proteins was also demonstrated in vitro with fluoresceinated antibody-labelled worms in the presence of xenogeneic molecules. The implications of these results are discussed in relation to the mechanisms of parasite evasion and "self-cure" phenomenon.     

Schistosoma mansoni worms induce anergy of T cells via selective up-regulation of programmed death ligand 1 on macrophages

Infectious pathogens can selectively stimulate activation or suppression of T cells to facilitate their survival within humans. In this study we demonstrate that the trematode parasite Schistosoma mansoni has evolved with two distinct mechanisms to suppress T cell activation. During the initial 4- to 12-wk acute stages of a worm infection both CD4(+) and CD8(+) T cells are anergized. In contrast, infection with male and female worms induced T cell anergy at 4 wk, which was replaced after egg laying by T cell suppression via a known NO-dependent mechanism, that was detected for up to 40 wk after infection. Worm-induced anergy was mediated by splenic F4/80(+) macrophages (Mphi) via an IL-4-, IL-13-, IL-10-, TGF-beta-, and NO-independent, but cell contact-dependent, mechanism. F4/80(+) Mphi isolated from worm-infected mice were shown to induce anergy of naive T cells in vitro. Furthermore, naive Mphi exposed to live worms in vitro also induced anergy in naive T cells. Flow cytometry on in vivo and in vitro worm-modulated Mphi revealed that of the family of B7 costimulatory molecules, only programmed death ligand 1 (PD-L1) was selectively up-regulated. The addition of inhibitory mAb against PD-L1, but not PD-L2, to worm-modulated Mphi completely blocked the ability of these cells to anergize T cells. These data highlight a novel mechanism through which S. mansoni worms have usurped the natural function of PD-L1 to reduce T cell activation during early acute stages of infection before the subsequent emergence of egg-induced T cell suppression in the chronic stages of infection.     

Partial molecular characterization of Sm8, a tegumental antigen of Schistosoma mansoni

Sm8 is a major tegumental antigen of Schistosoma mansoni. The partial cDNA was isolated and analyzed. Sequence analysis revealed transmembrane compatible hydrophobic domains and a putative leucine zipper pattern. The mRNA and the protein are predominantly expressed in adult worms.     

Scanning electron microscopical studies on the tegument of adult worms of Schistosoma mansoni originating from ultraviolet-irradiated and non-irradiated cercariae.

The surface topography and ultrastructural changes of Schistosoma mansoni worms developed from ultraviolet-irradiated cercariae were compared with those from non-irradiated cercariae. The tegument of worms developed from irradiated cercariae showed a variety of changes including the occurrence of oedemata and most of the tubercles being torn at the tip and having lost their spines on the dorsal surface of some male worms. In females, parts of the tegument were devoid of spines and surface lesions were present. The range and extent of these changes differed not only among individual worms but also between different regions of individual worms resulting in generalized deformities in those worms originating from irradiated cercariae.     

Schistosoma japonicum is less sensitive to cyclosporin A in vivo than Schistosoma mansoni.

We compared the toxic effects of cyclosporin A (CsA) on postmigratory immature Schistosoma mansoni and Schistosoma japonicum in mice. For each species, CsA was administered either relatively early or late during development. Exposure of 20-day-old S. mansoni to 1 subcutaneous dose of CsA (50 mg/kg) reduced the worm burden by 45% and induced herniae and/or boli of the gut in 32% of perfusable worms. These results agree with previous reports. In addition, CsA induced a marked liver shift (37% of total worm number). For S. japonicum, CsA was administered at 11 days postinfection (PI) because this species migrates more quickly. Killing of worms and damage to the gut were not observed, and only a slight liver shift occurred. Similarly, these effects were not recorded when CsA was administered at the later times of 34 days PI for S. mansoni and 17 days PI for S. japonicum. For both species, CsA stunted worms, affecting both sexes early PI but only females late PI. In conclusion, immature worms of S. japonicum are less sensitive than S. mansoni to CsA. Also, S. mansoni displays marked age-dependent differences in its sensitivity to CsA.     

Functional analysis of a T cell line specific for antiidiotypic antibodies to a Schistosoma mansoni protective epitope. II. Induction of protective immunity in experimental rat schistosomiasis.

In our previous work on the idiotypic network in the rat model of schistosomiasis we showed that immunization with an IgE mAb specific for 26/56-kDa parasitic Ag resulted in the production of anti-anti-Id antibodies of both the IgG and IgE classes. Further studies demonstrated that anti-Ab2 T cell lines, obtained by immunization with Ab2 antibodies, functioned as conventional Th cells; they were MHC-restricted and required APC to proliferate in the presence of the native schistosomula Ag and the Ab2 antibodies. We report the involvement of these anti-Ab2 cells in the regulation of protective immunity. The transfer of long term culture anti-Ab2 T cell lines into LOU/M rats, followed by a challenge infection by Schistosoma mansoni 1 day after the cell transfer led to a slight increase in the worm burden. On the contrary, the transfer of anti-Ab2 T cells 90 days before S. mansoni infection induced a significant reduction of the worm burden (up to 57%). T cells recovered from the protected rats were stimulated by the native schistosomula Ag as well as by tryptic fragments of IgG isolated from the Ab2 sera, in the presence of irradiated thymic cells as APC. We also analyzed the humoral response developed by the rats after transfer with the anti-Ab2 T cell lines. The sera induced various inflammatory cells into cytotoxic effectors against the larvae of S. mansoni, arguing for the presence of functional IgE in the sera. Moreover, when these sera were passively transferred into rats infected 1 day later, a significant reduction of the worm burden was observed. However, antibody-dependent cytotoxic mechanisms efficient 10 days after the anti-Ab2 T cell transfer did not correlate with the protective immunity which required a 90-day delay to be established. These data suggest that the protective immunity induced by the anti-Ab2 cells is supported both by the cellular and humoral components and that in a future vaccinating strategy the idiotypic network may play a crucial role.     

Cloning and gene expression of Schistosoma mansoni protease

Schistosomes utilize proteases (termed hemoglobinases) for degradation of host globin. cDNA clones encoding Schistosoma mansoni protease were isolated by immunologically screening an expression cDNA library with antisera raised against purified hemoglobinase. Confirmation of the identities of the clones was obtained immunologically and biochemically. The bacterially produced fusion protein encoded by one clone, lambda Hb2, degraded hemoglobin in vitro. The sequence of this clone suggested that this S. mansoni protease is synthesized in a precursor form in vivo. Gene titrations indicated that S. mansoni contains multiple genes corresponding to this cDNA. The expression of these genes may be regulated during the organism's life cycle since adult, female worms contained the highest abundances of homologous mRNA and protein compared to other stages.     

Tegumental changes in adult Schistosoma mansoni harbored in mice treated with artemether

A detailed temporal examination was made of alterations induced by artemether in the tegument of adult Schistosoma mansoni worms using scanning electron microscopy (SEM). Mice infected with S. mansoni cercariae 42 days previously were treated intragastrically with artemether at a single dose of 400 mg/kg. Groups of 3 mice were killed at 24 hr, 72 hr, and 7 days after treatment; the worms were collected by perfusion and examined by SEM. Twenty-four hours after artemether treatment, focal damage to the tubercles on the tegumental surface of male worms was seen. In both male and female worms, there was focal swelling and fusion of tegumental ridges, and sometimes peeling. After 72 hr, the damage to the tegument had increased, especially in female worms, with extensive swelling, fusion, and peeling of the tegumental ridges. In the most severely damaged worms, host leukocytes were seen to be adhered to the damaged tegument. Damage to the oral sucker was also occasionally seen in both male and female worms. Seven days after treatment, the appearance of the tegument had returned to normal in some male and female worms, whereas others still showed apparent damage. The results demonstrate that artemether damages the tegument of adult S. mansoni, and the intensity of damage is more severe in female worms than in males.     

Adult worm tegumental damage and egg-granulomas in praziquantel-resistant and -susceptible Schistosoma mansoni treated in vivo

The effect of treatment with praziquantel (PZQ) on the tegument of adult Schistosoma mansoni worms and on liver egg-granulomas has been examined in mice infected with PZQ-resistant and -susceptible parasite isolates. Two PZQ-resistant S. mansoni isolates, one selected by passage in the laboratory under drug pressure and one from Senegal established from eggs excreted by an uncured patient, were compared with PZQ-susceptible control isolates. Scanning electron microscopic observations on the tegument of Schistosoma adult worms treated in vivo with PZQ showed that more severe damage was inflicted by PZQ on susceptible worms than on drug-resistant worms. Observations on the pathology of Schistosoma egg-granulomas in the liver of infected mice after treatment with PZQ indicated that eggs from susceptible control isolates were more sensitive to PZQ than those from drug-resistant isolates.     

Nonenzymatic isolation of Trichinella pseudospiralis infective L1 larvae at pH 7.4

Over half of the number of Trichinella pseudospiralis infective L1 larvae recovered from host carcasses by pepsin-HCl digestion were isolated from homogenized carcasses incubated in HBSS. More worms isolated by the latter method were viable compared to those isolated by pepsin-HCl digestion. When host carcasses infected with T. pseudospiralis were diced into pieces and incubated in HBSS, 30% more worms were recovered than from homogenized carcasses incubated in HBSS as above, and the majority of worms acquired by the former method were viable. The infectivity of T. pseudospiralis infective L1 larvae isolated from homogenized muscle in HBSS was 3.9 times greater than that for larvae recovered from homogenized carcasses by pepsin-HCl digestion. Only 4% and 0.8% of the number of T. spiralis recovered from homogenized muscle by pepsin-HCl digestion were isolated from homogenized or diced muscle incubated in HBSS, respectively. Fewer T. spiralis isolated from homogenized tissue in HBSS were viable compared to those recovered from homogenized carcasses digested in pepsin-HCl or diced carcasses incubated in HBSS.     

The fecundity of Schistosoma mansoni in chronic nonhuman primate infections and after transplantation into naive hosts

Adult Schistosoma mansoni worms were transplanted from 8 nonhuman primates with chronic infections into 8 naive recipients, in an effort to test the hypothesis that worm fecundity reduction in chronic infections is the result of host immunity or some other host effect. Techniques for perfusing living donors without the added use of anti-schistosomal drugs and for reducing the likelihood of post-operative bacterial endotoxemia and septic shock are described. Fecundity values in terms of eggs per day per female worm were obtained for the worms in their original and in their new hosts and compared. In 3 experiments, perfusions were incomplete and the donors were saved, enabling direct comparisons of fecundity to be made in subpopulations of worms in both their original and new hosts, after equal life spans. In only 1 of the 8 transplantations was there a clear increase in fecundity after surgical introduction into a naive host. Therefore, these experiments fail to support the hypothesis that reduced fecundity of S. mansoni worms in permissive nonhuman primate hosts is a reversible result of host immunity or some other host-derived factor. Despite this negation, further evidence for reduced worm fecundity in older infections was obtained. In the absence of in vivo evidence for immune-mediated antifecundity, worm senescence is the most likely explanation for this finding, with irreversible immune damage to the worms being a less attractive alternative hypothesis.     

Alterations in the longevity and fecundity of adult Trichinella pseudospiralis related to method of isolation of infective larvae

The infectivity of Trichinella pseudospiralis infective larvae was reduced significantly following exposure to low pH or a combination of 1% pepsin at low pH compared to that for larvae isolated in phosphate-buffered saline (PBS) at pH 7.0. Reduction of host gastric pH by administration to mice of sodium bicarbonate solution in PBS was accompanied by an increase in the infectivity of larvae isolated in 1% pepsin/HCl (P/HCl) compared to that for worms inoculated into hosts given PBS alone. Fewer adult worms developing from larvae isolated in P/HCl became established in the host small bowel than was seen with larvae isolated in PBS; moreover, the fecundity in vitro of adult worms developing from P/HCl-isolated larvae was reduced below that for adults developing from larvae isolated from host muscle in PBS. More adult worms were recovered following infection of immune hosts with PBS-isolated larvae than were recovered from immune mice challenged with larvae isolated in P/HCl. Similar findings were observed in mice immunized by infection with Trichinella spiralis and challenged with T. pseudospiralis larvae isolated in either P/HCl or PBS. Immunization of mice with T. pseudospiralis larvae isolated by either method and challenged with larvae of T. spiralis resulted in recovery of similar percentages of the challenge inoculum.     

Schistosoma mansoni: stage-dependent formation and repair of membrane pores induced by a cytotoxin from Pseudomonas aeruginosa.

Various stages of Schistosoma mansoni were treated with a cytotoxin from Pseudomonas aeruginosa and their response to the damaging effect was studied in detail. Marker release and membrane potential measurements showed that the cytotoxin formed stable pores in all developmental stages. However, in juvenile 27-day-old worms, which are refractory to the killing effect of the cytotoxin, the pores had a smaller functional diameter as compared to other stages including 31-day-old worms. Furthermore, these resistant 27-day-old worms, but not susceptible older juvenile worms were able to repair the membrane lesions as shown by restoration of the resting membrane potential. In contrast, older juvenile and adult parasites were unable to cope with the breakdown of the resting potential induced by the cytotoxin. The results demonstrate the existence in 27-day-old schistosomes of effective repair mechanisms dealing with damage to the surface membrane.     

Effects of low-protein diet on Schistosoma mansoni morphology visualized by morphometry and confocal laser scanning microscopy

Previous studies have shown that protein deficiencies can hamper both the course of experimental schistosomiasis and normal development of adult worms. To further investigate this relationship, we compared adult male and female Schistosoma mansoni from malnourished and well-fed mice through morphometric and confocal laser scanning microscopy analysis. Swiss mice were fed protein-deficient diets (8%) and infected subcutaneously with approximately 80 S. mansoni cercariae (BH strain, Brazil). Control mice were fed a standard rodent diet (23% protein). The nutritional status was evaluated by body weight gain and albumin values. Mice were sacrificed 63 days post-infection. Recovered worms were stained with hydrochloric carmine and preserved as whole-mounts for bright-field examination and confocal microscopy. The body weight gain and serum albumin concentrations were significantly lower (P < 0.05) in malnourished mice than in controls. In general, all morphometric values of specimens grown in malnourished mice were lower than those of control mice. Schistosome worms grown in malnourished mice had statistically significant differences (P < 0.05) in the reproductive system and tegument than those grown in mice fed standard diets. In female worms, vitelline glands showed few remaining follicles and ovaries lacked mature oocytes. In male parasites, tubercles were fewer in number on the dorsal surface and testicular lobes presented fewer differentiated germinal cells. In summary, we describe novel data supporting the view that low-protein diets may influence the development of adult worms.     

The hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni. Further characterization and gene expression in Escherichia coli

Due to the lack of de novo purine nucleotide biosynthesis, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential enzyme in the human parasite Schistosoma mansoni for supplying guanine nucleotides and has been proposed as a potential target for antiparasitic chemotherapy. While the enzyme can be purified from adult schistosome worms, yields are too low to allow extensive structural and kinetic studies. We therefore cloned and sequenced the cDNA and gene encoding the schistosomal enzyme but were unable to positively identify the amino-terminal sequence of the enzyme from the DNA sequence. Knowledge of the exact amino terminus was necessary before accurate expression of active enzyme could be attempted. Therefore, we purified the HGPRTase from crude extracts of the adult worms. The purified enzyme has a subunit molecular mass of 26 kDa and an amino-terminal sequence of Met-Ser-Ser-Asn-Met. This sequence matched one of the potential initiation sites predicted from the cDNA and gene sequence. We next expressed the correct size cDNA of the S. mansoni HGPRTase in Escherichia coli using a vector that is regulated by a bacterial alkaline phosphatase promoter and uses an E. coli signal peptide for secretion of expressed product into the periplasmic space. Using this expression system, some of the recombinant enzyme is secreted and found to have a correct amino terminus. That remaining in the cytoplasm has part of the signal peptide attached to the amino terminus. The recombinant schistosomal HGPRTase isolated from the periplasm of the transformed E. coli was purified and found to have kinetic and physical properties identical to those of the native enzyme.     

Autoradiographic analysis of Schistosoma mansoni migration in the NZ rabbit

The migration of larval Schistosoma mansoni was tracked by means of autoradiographic analysis in naive rabbits percutaneously exposed to L-(75Se) selenomethionine-labeled cercariae on serial intervals of 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 40 and 50 days post-infection. Autoradiographic foci were detected from the 1st day in the skin, up to the 15th day in the liver. Adult and mature worms were recovered either paired or not 60 days after infection, by perfusion of hepatic and mesenteric veins. Morphometric analysis under optical microscopy, showed that worms were within regular dimention limits as compared to adult worms harboured by other host species. These observations extend previous informations on the S. mansoni-rabbit association and clearly demonstrate the post-liver phase of S. mansoni life-cycle in this host.     

A monoclonal antibody from infected mice to a Schistosoma mansoni egg proteinase

A number of monoclonal antibodies were obtained by fusion of SP2/0 myeloma cells and spleen lymphocytes from mice infected with Schistosoma mansoni. These antibodies were tested for their ability to inhibit acidic, thiol-dependent proteinases previously isolated from Schistosoma mansoni eggs and adult worms. One of the monoclonal antibodies isolated inhibits egg proteinase activity measured in vitro with the use of a low m.w. synthetic substrate. This antibody, which is an IgG1 isotype, does not appreciably inhibit an acidic, thiol-dependent proteinase obtained from the adult stage of Schistosoma mansoni. Immunocytochemical methods with the monoclonal antibody have been used to localize the egg proteinase within a set of "penetration" glands in the unhatched miracidium.     

Eimeria nieschulzi and Trichinella spiralis: Analysis of concurrent infection in the rat

The effects of concurrent primary infection of the rat with Eimeria nieschulzi and Trichinella spiralis on the number of oocysts of E. nieschulzi shed by the host and on the number, distribution, and fecundity of adult T. spiralis were analyzed. When rats were initially infected with E. nieschulzi followed 9 days later by infection with T. spiralis there occurred a significant decrease in the total numbers of adult worms in the small intestine, a significant shift in the position of these worms along the length of the small gut, a decrease in the fecundity of adult female worms, and a decrease in muscle parasitism when compared with rats infected with T. spiralis alone. When rats were initially infected with T. spiralis, followed 9 days later by infection with E. nieschulzi, there occurred a significant decrease in the numbers of oocysts shed over 24 hr on Days 7, 9, and 11 postinfection below that seen with rats infected only with Eimeria. These changes are discussed in terms of the enteropathophysiologic lesions and enteric inflammation known to occur during infections with these two parasites.     

Distribution of schistosome infections in molluscan hosts at different levels of parasite prevalence

Biomphalaria glabrata snails infected with Schistosoma mansoni were collected during consecutive seasons from a site in Brazil known to have a very high percentage of infected snails. Schistosoma mansoni cercariae from single snails were used to infect individual mice, and the recovered adult worms were genetically assessed using a mtVNTR marker. The number of unique parasite genotypes found per snail was compared to expected abundance values, based on the infection prevalence at the site, to determine the distribution of S. mansoni infections within the snail population. The observed distributions and those from previous studies were used to examine the relationship between schistosome prevalence and aggregation across a wide range of prevalence values. Our analysis showed that prevalence was inversely related to the degree of parasite overdispersion, and at high prevalence, S. mansoni infections were randomly distributed among snails.     

Light and confocal microscopic observations of adult Schistosoma mansoni from mice fed on a high-fat diet

The morphological aspects of Schistosoma mansoni adult worms recovered from albino mice fed on a cholesterol-rich diet compared to mice fed on a standard chow were investigated. After feeding on their respective diets for over a period of 5 months, mice were subcutaneously infected with c. 50 S. mansoni cercariae/mouse. Blood samples were obtained 1 day prior to experimental infections and 63 days later, when mice were euthanized by jugular section (hypovolaemic shock). Total cholesterol (TC) levels were determined. Recovered worms were stained with hydrochloric carmine, and preserved as whole-mounts for examination by bright-field and laser confocal microscopy. The infected mice fed on high-fat chow showed higher levels of serum lipoproteins than the infected mice fed on standard chow, except for very-low-density lipoprotein cholesterol (VLDL-c) and triglycerides (TG). In this experiment, worms from mice fed on a high-fat chow showed a greater percentage of morphological differentiation regarding supernumerary testes, seminal vesicle, and seminal receptacle. In mice of this group, the rate of oocyte laying in the ovary was much higher than in control females. The present results suggest that cholesterol could be actively involved in the modulation of cell signalling and reproduction, because the lobes contained fully developed oocytes in variable amounts, different from control males. The data presented here are the first to report the role of a cholesterol-rich diet affecting the development of S. mansoni worms.