Evaluation of the anthelmintic effects of artesunate against experimental Schistosoma mansoni infection in mice using different treatment protocols

The therapeutic effects of artesunate against experimental Schistosoma mansoni infection in mice were analyzed. Previous studies showed that artesunate is highly effective against S. japonicum infection, but the action of this drug against S. mansoni remained uncovered. The present study examines the optical conditions for artesunate against S. mansoni and evaluates the effects of inhibiting the sexual maturation of adult worms. Mice infected with S. mansoni were orally administered with artesunate according to different schedules. Four consecutive administrations of 300 mg/kg of artesunate at 2-week intervals conferred almost total protection without the development of pathological lesions in the liver. The significant reduction in the number of eggs produced by surviving worms and the status of egg maturation suggested that artesunate inhibits sexual maturation. Electron microscopy revealed that artesunate caused morphological damage, especially on the worm tegument. Artesunate was also very effective in iron-deficient mice. Furthermore, the efficacy of artesunate was equal to or better than that of artemether against S. japonicum infection. Considering that artemether is more toxic, artesunate is currently one of the most efficient drugs against immature S. mansoni.     

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.     

Schistosoma japonicum: the pathology of experimental infection

The pathology of experimental schistosomiasis japonica is reviewed and compared with the pathology of schistosomiasis japonica in man and to some aspects of schistosomiasis mansoni and schistosomiasis haematobia in experimental animals. The induction of granulomas around Schistosoma japonicum eggs depends upon cell mediated immunity, as do the reactions to Schistosoma mansoni and Schistosoma haematobium eggs. However, the modulation of the reaction to S. japonicum eggs can be greatly influenced by antibody, while antibody has no effect on the granulomas around S. mansoni eggs. Adult worm pairs of S. japonicum tend to cluster in the mesenteric venules, and most eggs are laid in a few sites. This leads to large, focal intestinal lesions similar to the discrete lesions produced by S. haematobium in the intestine and urinary tract but in contrast to the widespread, diffuse lesions produced by S. mansoni. Comparison with S. japonicum infection in humans is limited chiefly by our scant knowledge of the pathology produced by S. japonicum in infected persons. Most such comparisons are, in any case, limited by the marked differences in the reactions of various experimental host species to the infection and by differences in the reaction of a given host species to different strains of the parasite.     

A purified 28,000 dalton protein from Schistosoma mansoni adult worms protects rats and mice against experimental schistosomiasis

We have purified a 28,000 dalton (P28) protein from Schistosoma mansoni adult worms and used it to immunize Fischer rats. Immunofluorescence assays demonstrated that the P28 antigen was mainly located in the parenchyma of the schistosomulum and of the adult worm, including the dorsal spines of the parasite. Western blot analysis revealed that this antigen was present in three species of schistosomes: S. mansoni, S. japonicum, and S. bovis. The antibody response raised against this protein was able to kill S. mansoni schistosomula in in vitro cytotoxicity assays in the presence of rat eosinophils. The inhibition of this cytotoxic activity by an aggregated myeloma IgG2a indicated that one of the major isotypes involved in this in vitro model is IgG2a. The passive transfer of P28 antisera induced a significant level of protection against experimental infection. Moreover, we have immunized Fischer rats and BALB/c mice with the purified 28,000 dalton protein and observed a marked decrease (up to 70%) in the parasite burden in both experimental infection models.     

Worm morphology of Schistosoma japonicum using confocal laser scanning microscopy

Male and female Schistosoma japonicum worms have dissimilar appearances in their final host. In this study, a morphometric and morphological assessment of whole worms derived from unisexual and mixed infections in mice was conducted using confocal laser scanning microscopy. Worms from mixed infections showed significant morphological changes between 15 and 25 days post-infection (PI). On the fifteenth day PI, 33% of males had formed the conspicuous gynecophoric canal, but only 8% of them had testicular lobes containing a few germinative cells; 13% of females had incipient ovaries with a few immature ovarian cells inside. On the twentieth day PI, the testicular lobes contained more germinative cells in all male worms, while female worms presented vitelline glands. On the twenty-fifth day PI, more germinative cells were observed in the male testicular lobes, and differentiated cells were present in the female ovaries. All worms had fully developed reproductive organs from 30 days PI onwards. Morphometric analysis showed significant differences between mixed and unisexual infections at 35 days PI. Ovaries of worms from unisexual infections contained cells in one stage of maturation and vitelline glands had undifferentiated cells. Our study of S. japonicum provides a detailed comparison of different morphological traits from worms of mixed and unisexual infections throughout development.     

Schistosoma haematobium (Egyptian strain): rate of development and effect of praziquantel treatment

This study investigates the development of the Egyptian strain of Schistosoma haematobium and the resultant immunohistopathology and biochemical changes in organs affected. In addition, the response of different developmental stages of S. haematobium worms to praziquantel (PZQ) was examined. Schistosoma haematobium-infected hamsters were classified into 4 groups and were treated at day 35, 55, 75, and 95 postinfection (PI), respectively. Each group was subdivided into 3 subgroups. Two of them were treated orally with PZQ (300 mg/kg or 500 mg/kg divided equally on 2 consecutive days), and the third group was left without treatment. Treated groups were killed 20 days posttreatment. Infection with S. haematobium became patent 73 days PI; tissue egg load and worm fecundity were higher at 95 days and maximal 115 days PI, with an oogram pattern comparable to that in Schistosoma mansoni infection. In the liver, small cellular granulomas were observed 75 days PI, with preponderance of CD4+ T-cell phenotypes. In the urinary bladder, only submucosal focal Brunn's-nest formation and angiogenesis without typical granulomas were observed. Ninety-five and 115 days PI, confluent granulomata with multiple eggs in the center were observed in the liver and urinary bladder, with a preponderance of CD8+ positive T cells in the liver and hyperplasia of the urinary bladder epithelium with cystitis cystica and papillae formation. One hundred percent worm eradication was recorded with the higher dose of PZQ in animals treated 75 and 95 days PI. In conclusion, in spite of the long prepatent period of the Egyptian strain of S. haematobium, sensitivity to PZQ was recorded soon after infection. Granulomata were similar to those of S. mansoni in the livers and urinary bladders, but they were confluent with multiple eggs in the centers, hyperplasia of the urinary bladder urothelium with cystitis cystica, papillae, and Brunn's-nest formation predictive of malignant changes with no hepatocyte dysplasia.     

Immunization of mice with cells from juvenile worms of Schistosoma japonicum provides immunoprotection against schistosomiasis

To validate the protective efficacy against schistosomiasis by immunization with cells from juvenile Schistosoma japonicum in a murine model and to analyze possible factors related to protection, in this study, two independent repeated vaccination trials were performed. After three subcutaneous vaccina- tions, in trial one, in the absence of adjuvant, primary juvenile worm cells (pJCs) from S. japonicum induced remarkable average reductions in worm burden (54.3%), liver eggs per gram (LEPG) load (59.8%) as well as egg granulomas size (66.5%) compared to PBS control group (P<0.01), which were significantly higher than those elicited by fractions of juvenile worm cells (JCFs) or fractions of juvenile worms (JWFs) (P<0.05). Non-cell components of worms (WNCs) showed no significant protection. In trial two, compared to PBS control group, significant protective effect was also observed for cultured juvenile worm cells (cJCs) from S. japonicum with 58.4% worm reduction and 68.1% LEPG reduction (P<0.01). However, cultured adult worms cells (cACs) showed significantly higher worm burden (P<0.05) and egg burden (P<0.01) when compared to cJCs. Immunological analysis of trial two revealed that cJCs engendered a Th1-biased mixed Th1/Th2 type of immune response while cACs elicited a Th2-type response. Our data indicated that immunization with both primary and cultured cells from S. japonicum juvenile worms provided high immunoprotection, for which the physical character of immunogens, stage-specific parasite and the type of immune response induced might be responsible, suggesting that vaccination with whole cells from S. japonicum larvae is a promising approach to produce protec- tive immunity against schistosomiasis.     

Evidence for the presence of active cytochrome P450 systems in Schistosoma mansoni and Schistosoma haematobium adult worms

Extracts of the adult worms of both Schistosoma mansoni and Schistosoma haematobium can metabolise some typical P450 substrates but to differing degrees. S. mansoni worm extracts displayed a approximately 12-fold higher specific activity for an aminopyrine substrate than rat liver microsomes. At 4 mM substrate concentration the demethylation reaction with N-nitrosodimethylamine (NDMA) (5 nmol HCHO/mg protein/min) was only half that of rat liver microsomes, whereas in extracts of S. haematobium, no detectable activity was found towards NDMA. Using ethylmorphine as substrate the demethylation activity of S. mansoni extracts (1.82 nmol HCHO/mg protein/min) was 5.5-fold lower than that of rat liver microsomes. Benzphetamine demethylase activity was also readily detectable in S. mansoni worm extracts at 6.79 nmol HCHO/mg protein/min compared with 10.20 nmol HCHO/mg protein/min in the case of rat liver microsomes. When aniline was used as substrate, surprisingly, no activity was found in worm extracts of either S. mansoni or S. haematobium, whereas rat liver microsomes showed high activity towards this amine. The anti-P450 2E1 and 2B1/2 cross-reacted with both worm homogenates and gave a specific band corresponding to a protein of molecular weight of approximately 50.0 kDa. A study with anti-P450 IVA antibody revealed that while this protein was strongly expressed in S. haematobium worm extracts, no immunoreactivity was observed with extracts of S. mansoni. Immunoblotting analyses with anti-P450 IIIA and P450 1A1 did not detect immunoreactive protein in either S. mansoni or S. haematobium.     

Schistosoma malayensis n. sp.: a Schistosoma japonicum-complex schistosome from Peninsular Malaysia

Schistosoma malayensis n. sp., a member of the Schistosoma japonicum complex is described from Rattus muelleri in Peninsular Malaysia and 2 strains are characterized. The only morphological differences noted among adults from natural hosts were that S. malayensis are in general smaller than S. mekongi and S. japonicum. But these differences may be the result of host-induced variations and therefore are of little taxonomic value. To minimize the effects of host-induced variations, adult worms recovered from laboratory mice with similar worm burdens at 50-56 days postinfection were compared. These comparisons revealed only minor morphometric differences among these 3 species. Schistosoma malayensis eggs from naturally and experimentally infected hosts are most similar to those of S. mekongi, with eggs of both species being, in general, smaller than those of S. japonicum. The egg index for S. malayensis is usually higher than for S. japonicum and lower than for S. mekongi. Differences were noted in the developmental rates in mice for 2 isolates of S. malayensis, S. mekongi, and S. japonicum (Philippine strain), but relatively large differences observed between isolates of S. malayensis indicate that, in this case, the developmental rate is not a useful taxonomic character. Schistosoma malayensis is erected principally on the basis of differences, reported elsewhere, in the life histories and in the electrophoretic migration patterns of isoenzymes of adult worms as compared to S. mekongi and S. japonicum. These comparisons indicate that S. malayensis is more closely related to S. mekongi than to S. japonicum.     

Structure and bioactivity of neuropeptide F from the human parasites Schistosoma mansoni and Schistosoma japonicum

The blood flukes Schistosoma mansoni and Schistosoma japonicum inflict immense suffering as agents of human schistosomiasis. Previous investigations have found the nervous systems of these worms contain abundant immunoreactivity to antisera targeting invertebrate neuropeptide Fs (NPFs) as well as structurally similar neuropeptides of the mammalian neuropeptide Y (NPY) family. Here, cDNAs encoding NPF in these worms were identified, and the mature neuropeptides from the two species differed by only a single amino acid. Both neuropeptides feature the characteristics common among NPFs; they are 36 amino acids long with a carboxyl-terminal Gly-Arg-X-Arg-Phe-amide and Tyr residues at positions 10 and 17 from the carboxyl terminus. Synthetic S. mansoni NPF potently inhibits the forskolin-stimulated accumulation of cAMP in worm homogenates, with significant effects at 10(-11) m. This is the first demonstration of an endogenous inhibition of cAMP by an NPF, and because this is the predominant pathway associated with vertebrate NPY family peptides, it demonstrates a conservation of downstream signaling pathways used by NPFs and NPY peptides.     

Rodents as reservoir hosts in the transmission of Schistosoma mansoni in Richard-Toll, Senegal, West Africa

More than 2000 animals belonging to six different rodent species and one insectivore species were examined for infection with schistosomes in the region of Richard-Toll, Senegal. Two murid rodents, Arvicanthis niloticus and Mastomys huberti, were found infected with Schistosoma mansoni. Prevalences were about 5% for both rodent species with a mean worm burden of about 20 worms per host. The sex-ratios of S. mansoni worms were always biased towards males. Prevalences and worm burdens, although similar in both male and female rodents, increased significantly with age. The highest prevalences and worm burdens were found near habitations and decreased significantly with the distance from the town of Richard-Toll. Eggs were also observed in the liver and faeces of the two naturally infected rodent species. The results suggest that rodents participate in the transmission of intestinal schistosomiasis in Richard-Toll but the human population is the main source of infection. The genetic resemblance between human and murine isolates of S. mansoni suggests that further epidemiological studies are needed in this region of Senegal.     

Isozyme patterns of Schistosoma japonicum and S. mansoni

Isozyme patterns of six enzymes, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, hexokinase, malate dehydrogenase, 6-phosphate dehydrogenase and phosphoglucomutase were examined in electrophoresed homogenates of adult male worms of Schistosoma japonicum and S. mansoni. In general, enzyme patterns obtained from the parasite homogenates differed from that of host (mouse) blood and muscle, indicating that electrophoretic patterns from parasite extracts are most probably of parasite origin. Adult male and female S. mansoni worms yielded identical patterns. However, all six enzyme patterns showed distinct differences between S. japonicum and S. mansoni. These results suggest that S. japonicum is clearly distinguishable from S. mansoni at the molecular level.     

Schistosoma japonicum: immunological characterization and detection of circulating polysaccharide antigens from adult worms

The antigenic constituents of a trichloroacetic acid (TCA)-soluble fraction of adult Schistosoma japonicum were studied with immunoelectrophoresis, and compared with those of Schistosoma mansoni. Eight TCA-soluble antigens of S. japonicum were demonstrated, five of which showed immunological identity with S. mansoni antigens. Of the eight antigens, five antigens with anodic motility were found as circulating antigens in S. japonicum-infected hamster and rabbit sera; the major circulating antigen was the circulating anodic antigen (CAA). Two other antigens, with cathodic motility, including the circulating cathodic antigen (CCA), were demonstrable as circulating antigens in S. mansoni infections, but not in S. japonicum infections. Most of the circulating antigens were shown to be gut-associated. Only one antigen, line 2, which was not demonstrable as circulating antigen and which was present in the parenchyma of the worms, was found to be specific for S. japonicum. Using an ELISA for the detection of CAA in the sera of S. japonicum-infected rabbits, a lower detection level of 100 ng CAA/ml serum was achieved. Moreover, at 7-8 weeks after infection, a direct relationship between worm burden and CAA level was demonstrated.     

日本血吸虫中国大陆株26kD GST基因在大肠杆菌中的高效表达

用大肠杆菌高效表达日本血吸虫中国大陆株 2 6k D GST基因 (Sj2 6)并观察表达产物诱导的免疫保护效果 .将 Sj2 6基因亚克隆至 p ET2 8b(+)中构建重组表达质粒 Sj2 6/ p ET,转化大肠杆菌 BL 2 1 (DE3) .Sj2 6在诱导条件下及未诱导条件下均获得高效表达 .表达产物 (r Sj2 6GST)以包含体形式表达 ,分子量为 2 8k D左右 ,能用 6× His亲和层析柱纯化 ,纯化产量为 55～ 60 mg/ L大肠杆菌培养物 .免疫印迹及 ELSA实验证实 r Sj2 6GST具有良好抗原性 .用 r Sj2 6GST不加佐剂直接背部皮下免疫昆明系小鼠 ,获得了 1 9.6% (P<0 .0 5)的减虫率及 31 .9% (P<0 .0 5)的成熟虫体减虫率 ;且免疫组检获的虫体中未成熟虫体的比例为 32 % (66/ 2 0 6) ,明显高于对照组的 1 9.6% (56/2 85) (P<0 .0 1 ) ,说明 r Sj2 6GST免疫不仅可诱导抗日本血吸虫的部分攻击感染作用 ,而且能抑制部分虫体的发育 .     

The ability of single sex infections of Schistosoma japonicum to induce resistance to reinfection in mice

In four experiments, mice harbouring an average 50, 76 or over 200 Schistosoma japonicum female worms were not resistant when challenged six to eight weeks after infection. The female worms from these single sex infections were stunted and immature (average length 4.6 mm) and induced no overt pathology in the host. Male worm burdens of 60, 135 or 140 also induced little or no resistance to challenge in the host. The males from these single sex infections were fully grown for their age (average length 9 mm) and burdens of 135 or 140 induced distended hepatic portal veins and marked deposition of pigment in the livers of infected mice.     

Schistosoma mansoni: the effect of adrenalectomy on the murine model

Adrenal steroid hormones have been implicated, among others, as one of the most important host factors controlling the onset, establishment, and pathogenesis of schistosomiasis. They appear to inhibit oviposition by Schistosoma mansoni both in vitro and in vivo, and their effect is greatly enhanced when administered in combination with a schistosomicidal drug. Therefore, we hypothesized that adrenalectomy would greatly affect the course of the murine schistosomiasis infection. To test this hypothesis, adrenalectomized mice (Adx) infected with S. mansoni were compared with intact infected and sham-infected controls concerning their mortality rate, numbers of male and female worms, number of eggs, and liver pathology. Compared with controls, Adx infected mice showed an increase of 50% in the mortality rate, as well as 1.7-3 times as many adult worms and twice as many ova per worm pair in their liver. Thus, for the first time, there is evidence that lack of adrenal steroids mediate an increment of the adult worm burden and promote worm fecundity in vivo. The present work was done to test the hypothesis that lack of adrenal steroids enhances adult worm attrition, possibly by their direct effect on the parasite, and by upregulating or downregulating innate and adaptive immune responses.     

Effect of artemether against Schistosoma haematobium in experimentally infected hamsters

The drug, artemether, has been shown to be active against the juvenile stages of Schistosoma japonicum and Schistosoma mansoni in experimentally infected animals, while it is less effective on adult worms. These findings have been confirmed in randomised controlled trials in humans. Consequently, it could be expected that artemether is also active against Schistosoma haematobium. We present here the first results from experiments assessing the effect of artemether on S. haematobium. Hamsters with a single infection received intra-gastrically an initial dose of 300 mg/kg artemether on day 14, 21 or 28, followed by further doses at varying treatment regimens. In all the treatment groups, the total and female worm reduction rates were highly significant, and ranged from 78 to 100% in hamsters harbouring juvenile schistosomes. Hamsters infected three times with S. haematobium, on days 0, 4 and 9, and repeatedly treated with artemether at the same dose as above, showed highly significant total and female worm reduction rates of between 94 and 99%. Artemether was also active against 77-day-old adult S. haematobium, since its administration on two consecutive days resulted in highly significant total and female worm reduction rates of 76-89%. Our findings confirm that artemether is also active against S. haematobium, especially the schistosomules. These results provide a basis for clinical trials in humans, for further assessment of the potential of artemether for schistosomiasis control.     

Leucine aminopeptidase of the human blood flukes, Schistosoma mansoni and Schistosoma japonicum

An array of schistosome endoproteases involved in the digestion of host hemoglobin to absorbable peptides has been described, but the exoprotease responsible for catabolising these peptides to amino acids has yet to be identified. By searching the public databases we found that Schistosoma mansoni and Schistosoma japonicum express a gene encoding a member of the M17 family of leucine aminopeptidases (LAPs). A functional recombinant S. mansoni LAP produced in insect cells shared biochemical properties, including pH optimum for activity, substrate specificity and reliance on metal cations for activity, with the major aminopeptidase activity in soluble extracts of adult worms. The pH range in which the enzyme functions and the lack of a signal peptide indicate that the enzyme functions intracellularly. Immunolocalisation studies showed that the S. mansoni LAP is synthesised in the gastrodermal cells surrounding the gut lumen. Accordingly, we propose that peptides generated in the lumen of the schistosome gut are absorbed into the gastrodermal cells and are cleaved by LAP to free amino acids before being distributed to the internal tissues of the parasite. Since LAP was also localised to the surface tegument it may play an additional role in surface membrane re-modelling.     

Ultrastructural alterations in adult Schistosoma mansoni caused by artemether

Progress has been made over the last decade with the development and clinical use of artemether as an agent against major human schistosome parasites. The tegument has been identified as a key target of artemether, implying detailed studies on ultrastructural damage induced by this compound. We performed a temporal examination, employing a transmission electron microscope to assess the pattern and extent of ultrastructural alterations in adult Schistosoma mansoni harboured in mice treated with a single dose of 400 mg/kg artemether. Eight hours post-treatment, damage to the tegument and subtegumental structures was seen. Tegumental alterations reached a peak 3 days after treatment and were characterized by swelling, fusion of distal cytoplasma, focal lysis of the tegumental matrix and vacuolisation. Tubercles and sensory organelles frequently degenerated or collapsed. Typical features of subtegumental alterations, including muscle fibres, syncytium and parenchyma tissues, were focal or extensive lysis, vacuolisation and degeneration of mitochondria. Severe alterations were also observed in gut epithelial cells and vitelline cells of female worms. Our findings of artemether-induced ultrastructural alterations in adult S. mansoni confirm previous results obtained with juvenile S. mansoni and S. japonicum of different ages.     

Lack of effect of unmated schistosomes on the fecundity of mated worm pairs

An imbalance between the numbers of male and female worms had no significant effect upon the fecundity of Schistosoma mansoni in rhesus monkeys or upon the number of S. mansoni eggs in the tissues of infected persons or mice. The number of S. japonicum eggs in the tissues of infected rabbits was similarly unaffected by disproportion between worm genders.