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.     

Effect of pairing in vitro on the glutathione level of male Schistosoma mansoni

The effect of in vitro incubation on the level of the intracellular nucleophile, glutathione (GSH), in adult Schistosoma mansoni was investigated. The GSH levels of freshly collected adult male and female parasites were 8.5 +/- 2.5 and 2.7 +/- 0.7 nmol/10 worms, respectively, as determined by an enzymatic assay. Twenty-four-hour incubation of unpaired males in RPMI-1640 medium at 37 C resulted in a 1.7-fold increase (P less than 0.001) in GSH level that remained elevated for at least 7 days. The increase was dependent on exogenous L-cystine, suggesting that it was due to biosynthesis of GSH. Biosynthesis in male S. mansoni was confirmed by isolating [3H] GSH from parasites incubated in medium containing L-[3H] cystine or [3H] glycine. In contrast to unpaired males, the GSH level of paired males as well as that of unpaired or paired females did not increase after 24 hr in vitro. When males that had been incubated unpaired for 24 hr were allowed to couple in vitro with freshly collected females, their GSH level fell to that of continuously paired males. These observations provide evidence that in vitro female schistosomes can influence the physiology of the male.     

The uptake, localization and transfer of [4-14C]cholesterol in Schistosoma mansoni males and females maintained in vitro

Schistosoma mansoni male and female adults incorporated [4-14C]cholesterol in vitro. Males incorporated about three times more cholesterol than females. The major sites of cholesterol deposition in males were the tegument and parenchyma. The tegument and vitelline glands were the primary sites of cholesterol accumulation in females. In males, dorsal tegument showed greater cholesterol uptake than ventral tegument. Radiolabelled males and females transferred cholesterol to unlabelled members of the opposite sex.     

Uptake of [14C]oxamniquine by Schistosoma mansoni

The uptake and retention of drug-related material by Schistosoma mansoni was studied in the mouse host following a single oral or intramuscular dose (50 mg/kg) of [14C]oxamniquine. Male worms took up more labelled material than did female worms but the amount in each particular sex of worm was found to be similar after both routes of administration. Exposure of worms was therefore independent of the route of administration. Six days after drug administration, at the time of an hepatic shift, significantly more drug-related material was present in male worms than in female worms. Examination of worms recovered from mice 4 h after treatment showed that metabolites of oxamniquine constituted 70--90% of the drug-related material present in the worms. Both sexes of worms were able to take up metabolites of oxamniquine in vitro.     

The role of Schistosoma mansoni males in feeding and development of female worms

Female Schistosoma mansoni from unisexual infections have scant pharyngeal musculature, thin intestinal cecal walls, pale and scanty intestinal contents, and lack acidic thiol proteinase digestive enzyme as determined by indirect immunofluorescence using a monoclonal antibody. Their intake of host erythrocytes, measured by 51Cr labeling, is about one-fourth that of paired adult females, and they appear to be starved. In contrast, paired adult females have heavier pharyngeal musculature and intestinal cecal walls and abundant digestive enzyme in the anterior third of their intestinal tract. Females in worm pairs surgically transplanted into uninfected mice continued to feed, but separated females were carried into the liver and deteriorated. Adult female S. mansoni, newly separated from their male partners and incubated in vitro with labeled erythrocytes, ingested marginally fewer cells than did still-paired females, indicating their ability to continue feeding almost normally at least for a period after separation. Paired and ex-paired adult females declined similarly in feeding rate with increased time in vitro. In Schistosomatium douthitti, females grow and mature without males, the pharyngeal musculature and cecal walls are well developed, the gut is full of ingested blood, and the acidic thiol proteinase is present in both unisexual and paired female worms. There are different stimulatory pathways for growth and for reproductive maturation in S. mansoni, although both processes require physical contact with the male. We believe that the growth-stimulating function results from the muscular action of the clasping male, which helps the immature female to pump blood into her intestine, thereby overcoming a state of relative starvation.     

Schistosoma mansoni: male-biased sex ratios in snails and mice

Adult sex ratios of Schistosoma mansoni, in mice, were shown to be biased toward males (3:1) despite the finding that sex ratios of miracidia were 50:50. The adult male bias was caused by greater male infectivity of miracidia for snails and cercariae for mice. A significantly higher percentage of male miracidia developed to cercarial production in unimiracidial infections (57 male, 34 female), and a significantly higher percentage of male cercariae developed to adulthood in mice (143 male, 79 female worms resulted from 900 male and 900 female cercariae). No significant differences were found between male and female parasites for longevity of miracidia (both sexes, 10 hr) and cercariae (males 21.3 +/- 5.75 hr, females 25.0 +/- 7.02 hr), prepatent periods in snail hosts (male 34 +/- 2.92 days, females 33 +/- 2.36 days), longevity of snail infections (males 96.6 +/- 25.15 days, females 115.2 +/- 82.43 days), and the numbers of cercariae produced per snail lifetime (males 30,751.44 +/- 18,064.33, females 34,083.00 +/- 33,732.82). Present results provide a better understanding of the life cycle of S. mansoni, are of theoretical significance for theories of biased sex ratios (which at present cannot account for the male-biased ratio of S. mansoni), and also suggest that schistosomiasis transmission models assuming a 50:50 sex ratio at all stages of the life cycle should be reassessed.     

Reproductive development of female Schistosoma mansoni (Digenea: Schistosomatidae) following bisexual pairing of worms and worm segments

Maturation and maintenance of normal reproductive function in female Schistosoma mansoni require a permanent association with the male, but the nature of this relationship is not well understood. The regional localization of a stimulatory factor in the male and its target in the female were investigated. Unisexual female and mature male worms were transected into segments of various lengths. Various combinations of transected male and female segments and intact worms were transferred to the mesenteric veins of recipient hamsters and were also maintained in vitro. In hamsters and in vitro, pairing took place between intact worms of each sex and segments of the other, and between segments of both sexes. The majority of female worms and segments so paired showed some reproductive development, as assessed by vitelline gland differentiation. In intact unisexual females paired with small male segments, vitelline gland development was limited to that portion of the worm that had been held by the male. Worm segments continued to display normal body contractions throughout 24 days of in vitro maintenance and morphological integrity was retained. It is concluded that 1) in the absence of a functioning gut, worm segments can survive for prolonged periods on nutrients absorbed through the tegument; 2) worm pairing, male stimulation, and the female developmental response are independent of central nervous control by the cerebral ganglia; 3) males have no centralized localization for the female-stimulating factor; 4) vitelline gland differentiation in the female requires local stimulation through male contact, and this is not propagated throughout the worm.     

In vitro and in vivo studies on the bioactivity of a ginger (Zingiber officinale) extract towards adult schistosomes and their egg production

The bioactivity of an ethyl acetate extract of ginger (Zingiber officinale) towards Schistosoma mansoni adult pairs, both cultured in vitro and in vivo in laboratory mice, was investigated by monitoring worm mortality and fecundity. In vitro, a concentration of 200 mg l(-1) of extract killed almost all worms within 24 h. Male worms seemed more susceptible than female under these conditions. Cumulative egg output of surviving worm pairs in vitro was considerably reduced when exposed to the extract. For example, after 4 days of exposure to 50 mg l(-1), cumulative egg output was only 0.38 eggs per worm pair compared with 36.35 for untreated worms. In vivo efficacy of the extract was tested by oral and subcutaneous delivery of 150 mg kg(-1) followed by assessment of worm survival and fecundity. Neither delivery route produced any significant reduction in worm numbers compared with untreated controls. Worm fecundity was assessed in vivo by cumulative egg counts per liver at 55 days post infection with mice treated subcutaneously. Such infections showed egg levels in the liver of about 2000 eggs per worm pair in 55 days, in both treated and control mice, with no significant difference between the two groups. To ensure that density-dependent effects did not confound this analysis, a separate experiment demonstrated no such influence on egg output per worm pair, at intensities between 1 and 23 worms per mouse.     

Schistosoma mansoni, S. japonicum, S. haematobium: glycogen content and glucose uptake in parasites from fasted and control hosts

The glycogen content of male and female Schistosoma mansoni has been measured in flukes from normally fed hosts and those from fasted hosts. In infections from both the mouse and the hamster, a significant reduction in schistosomal glycogen of males is seen hours after food is withdrawn from the host. Reductions in protein content of the schistosomes were only observed in hamster infections fasted at least 72 hr. The livers of infected mice not only decrease in size during fasting, but there is a concomitant reduction in glycogen per unit wet weight. Comparisons of glycogen:protein ratios of mansonian males, females, and host livers indicate that the fasting-induced loss of liver glycogen is also observed in the male schistosome, but not the female. Studies of both S. mansoni and S. haematobium pairs from fed hosts suggest that the ratio of glycogen:protein contents in the male schistosome correlates with the glycogen:protein ratio of the female partner. Measurements of glucose uptake in vitro suggest that greater uptake rates may be observed in flukes perfused from fasted hosts. In S. japonicum from infected mice, a reduction in male glycogen was also detected as early as after a 6-hr fasting period, but changes in the females were not significant. Unmated male S. japonicum also exhibit a reduction in glycogen levels after fasting, but the quantity of worm glycogen present in these males remains higher than comparable mated males. In mice entrained to a regulated pattern of available food, fluctuations in glycogen content of the male schistosomes were observed, but in the female partners fluctuations were of a smaller magnitude.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological aspects of Schistosoma mansoni adult worms isolated from nourished and undernourished mice: a comparative analysis by confocal laser scanning microscopy.

Malnutrition hampers the course of schistosomiasis mansoni infection just as normal growth of adult worms. A comparative morphometric study on adult specimens (male and female) recovered from undernourished (fed with a low protein diet - regional basic diet) and nourished (rodent commercial laboratory food, NUVILAB) white mice was performed. Tomographic images and morphometric analysis of the oral and ventral suckers, reproductive system and tegument were obtained by means of confocal laser scanning microscopy. Undernourished male specimens presented smaller morphometric values (length and width) of the reproductive system (first, third and last testicular lobes) and thickness of the tegument than controls. Besides that, it was demonstrated that the dorsal surface of the male worms bears large tubercles unevenly distributed, but kept grouped and flat. At the subtegumental region, vacuolated areas were detected. It was concluded that the inadequate nutritional status of the vertebrate host has a negative influence mainly in the reproductive system and topographical somatic development of male adult Schistosoma mansoni, inducing some alterations on the structure of the parasite.     

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.     

Schistosoma mansoni: cholesterol uptake by paired and unpaired worms

Mature males and females of Schistosoma mansoni were incubated for 24 hr in medium containing [3H]cholesterol. Worms thus labeled were paired for 24 hr with unlabeled partners, in vitro or in vivo by surgical implantation into hamsters. Controls consisted of additional unlabeled worms that did not pair. Scintillation counting of thin layer chromatographic separations of lipid extracts of schistosome tissues and of the culture medium indicated that [3H]cholesterol underwent no major metabolic changes during the course of the experiment. During the period of time allowed for pairing, labeled worms lost up to 65% of their [3H]cholesterol, which was detected in the pairing medium. In both unlabeled males and females which had paired with labeled partners, levels of [3H]cholesterol were higher than in unpaired controls. This suggests that normal cholesterol transfer in worm pairs is bidirectional and that it is facilitated by physical contact between juxtaposed membranes. Cholesterol exchange in schistosome worm pairs may be partly or wholly a consequence of normal tegumental turnover of the molecule.     

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.     

Hymenolepis diminuta: ultrastructural abnormalities in worms from C57 mice

Ultrastructural studies, including stereological analyses of micrographs, have been made of five-worm primary infections of Hymenolepis diminuta from C57 mice to determine whether the immune destrobilation/rejection process was accompanied by significant changes in the fine structure of the scolex tegument. Destrobilation/rejection of worms occurred from Days 9-12 after infection. For the first 5 days after infection, the scolex tegument showed no detectable differences in ultrastructure compared with that of "control" worms from either Wistar rats or immunosuppressed C57 mice. By Day 6, large lipid deposits were observed in the tegument and associated musculature of worms from untreated C57 mice. Further, worms recovered from Days 6-8 after infection also showed increased activity of the Golgi apparatus, GER, and mitochondria of the tegument, resulting in increased numbers of discoidal secretory bodies. Concomitant with destrobilation/rejection from Day 9 was a drop in the number of secretory bodies, an increase in autophagic activity throughout the tegument, and a blistering of the tegument surface plasma membrane. The possible functional significance of the results is discussed in relation to host immunity.     

New imidazolidinic bioisosters: potential candidates for antischistosomal drugs

The emergence of strains of Schistosoma resistant to praziquantel has drawn attention to the search for new schistosomacide drugs. Imidazolidinic derivatives have performed outstandingly against adult S. mansoni worms when evaluated in vitro. The molecular modification of imidazolidine by way of bioisosteric replacement gives rise to variations in its biological response. This study verifies the potential of substituent groups in the derivatives (Z)3-benzyl-5-(2-fluoro-benzylidene)-imidazolidine-2,4-dione NE4, 3-benzyl-5-(4-chloro-arylazo)-4-thioxo-imidazolidin -2-ona PT5, 3-benzyl-5-(3-fluoro-benzylidene)-1-methyl-2-thioxo-imidazolidin-4-one JT53; 3-benzyl-1-methyl-5-(4-methyl-benzylidene)-2-thioxo-imidazolidin-4-one JT63; 3-benzyl-1-methyl-5-(4-methoxi-benzylidene)-2-thioxo -imidazolidin-4-one JT68; 3-(4-chloro-benzyl)-1-methyl-5-(4-methoxi-benzylidene)-2-thioxo-imidazolidin-4-one JT69; 3-(4-phenyl-benzyl)-1-methyl-5-(4-methoxi-benzylidene)-2-thioxo-imidazolidin-4-one JT72 by determining the viability in vitro of adult S. mansoni worms in the presence of these derivatives. The susceptibility of the worms obtained from mice and kept in culture in the presence of different concentrations was determined by way of schistosomacide kinetic, observed every 24 h over a period of eight days. The results show that the worms were more sensitive to the PT5 derivative at a concentration of 58 microM which killed 100% of the worms after 24 h of contact, also giving rise to alterations in the tegument surface of the worms with the formation of bubbles and peeling. These observations suggest a strong electronic contribution of the arylazo grouping in the biological response.     

Cultivation of Clinostomum marginatum (Digenea: Clinostomatidae) metacercariae in vitro, in chick embryo and in mouse coelom

Metacercariae of Clinostomum marginatum obtained from naturally infected Perca flavescens were cultured by 5 methods. In vitro cultivation at 37 C in twice daily changes of Tyrode's, Na-poor Tyrode's, and Eagle's media failed to produce ovigerous adults after 4-5 days. Metacercariae placed on the chorioallantois of week-old chick embryos at 37 C migrated to the albumen. Only 8 of 123 worms recovered were ovigerous after 4-7 days in ovo. Best success occurred with metacercariae injected in groups of 4 or 6 into the abdominal cavities of male A/J mice. Despite liver lesions and strong inflammatory responses in the host, all 174 worms recovered were ovigerous after 4-8 days. None of 41 mice died prematurely from the procedure or the parasites.     

Detection of inducible nitric oxide synthase in Schistosoma japonicum and S. mansoni

Schistosoma japonicum and S. mansoni were tested for reactivity with an anti-inducible nitric oxide (iNOS) antibody and the distribution of iNOS was studied by immunofluorescent tests in different stages of the parasites. Reactivity was associated with the tegument in both larval schistosomes (sporocysts and cercariae) and eggs. With adult worms, the majority of the immunofluorescence was predominantly subtegumental in S. japonicum and parenchymal in S. mansoni. Fluorescence was also observed in host tissues (snails and mouse liver). In Western blots, the enzyme of S. japonicum had an apparent molecular weight of about 210 kDa. The possible role of worm and host iNOS in the parasite-host interrelation remains to be clarified.     

Hormonal and pharmacologic agents affecting choline uptake and incorporation in Schistosoma mansoni adults

Initial uptake of choline by Schistosoma mansoni (2-min uptake) revealed no differences between male, female or paired worms for any of the control or experimental groups. After a 30-min uptake period, however, males showed significantly higher uptake of choline in the presence of mitomycin C, cytochalasin B and calcium ionophore A23187, while paired worms showed significantly reduced uptake in the presence of actinomycin D, puromycin, mitomycin C, cytochalasin B, colchicine, insulin, thyroxine and lysine. Choline uptake by females was elevated, in the presence of cytochalasin B at 30 min, although not significantly. Significantly increased incorporation of choline into phosphatidylcholine was observed following a 30-min incubation with 5-hydroxytryptamine (males), puromycin and thyroxine (females) and calcium ionophore A23187 (males, females and pairs). These effects on phosphatidylcholine synthesis are discussed in relation to the uptake data and to previous work concerning the outer membrane complex of the parasite as an important facet of parasite resistance to the host immune response.     

Fasciola hepatica and Schistosoma mansoni: immunofluorescent antigen localization and cross-reactivity

In an attempt to identify the tissue sources of biochemically purified antigenic fractions of Fasciola hepatica and Schistosoma mansoni, antisera were tested against plastic-embedded sections of worms of various ages by an indirect fluorescent-antibody-labeling technique. Antibodies prepared against antigens purified by chromatography of F. hepatica whole worm extract through concanavalin A-Sepharose 4B labeled the parenchyma and tegument of adult F. hepatica strongly while antibodies developed against antigens purified by antibody-affinity chromatography against antibodies of S. mansoni labeled only the parenchyma. Antigens common to these two groups clearly originated from F. hepatica parenchyma. Certain of these common antigens are known to provide significant protection in mice to challenge with S. mansoni cercariae, and in the present study antisera against F. hepatica extracts cross-labeled S. mansoni adult male parenchyma. Reciprocal cross-reactions between antisera against S. mansoni and the parenchyma of adult F. hepatica were also noted. FhFIIb, an extract of F. hepatica which Tailliez described as not cross-reacting with S. mansoni, was found to contain no F. hepatica parenchymal antigens. Antigenic fractions of F. hepatica and S. mansoni collected from the surface of worms after incubation in nonionic detergent were unexpectedly found to contain much parenchymal antigen, suggesting leakage of internal components into the supernatant during preparation. Antisera to F. hepatica developed during a natural infection in rabbits labeled tegumental components and gut strongly but did not react with parenchymal tissue. Antisera against extracts of adult schistosomes labeled the parenchyma of male worms and the glycocalyx of the cercarial tegument, indicating the presence of common antigens in the adult and the cercarial stage. Reciprocal reactions between anticercarial sera and adult sections provided further evidence of shared antigenicity. Antisera against S. mansoni egg antigens strongly labeled sections of eggs in liver tissue and cross-reacted with cercarial glycocalyx, indicating the existence of common antigens between these two stages. The antisera also cross-reacted with what appeared to be non-membrane-bound protein in the tegument of F. hepatica. The soluble egg antigen extract shared antigenicity with the parenchyma of both S. mansoni and F. hepatica but circumoval precipitin had no cross-reactivity with this tissue. Thus S. mansoni eggs contain nondiffusable components sharing antigenic specificity with adult parenchymal tissue.(ABSTRACT TRUNCATED AT 400 WORDS)