Human serum activates the tegument of female schistosomes and supports recovery from Praziquantel.

Schistosomiasis is one of the most devastating parasitic disease in the world. Schistosoma spp. survive for decades within the vasculature of their human hosts. They have evolved a vast array of mechanisms to avoid the immune reaction of the host. Due to their sexual dimorphism, with the female worm lying within the gynecophoric canal of the male worm, it is the male that is exposed to the immediate environment and the soluble parts of the host's immune response. To understand how the worms are so successful in fending off the immune attacks of the host, comparative analyses of both worm sexes in human serum (with or without Praziquantel) were performed using scanning electron microscopy, transmission electron microscopy, and immunohistochemistry. Further, gene expression analyses of tegument-specific genes were performed. Following the incubation in human serum, males and females out of pairs show morphological changes such as an altered structure of the pits below the surface and an increased number of pits per area. In addition, female schistosomes presented a marked tuft-like repulsion of their opsonized surface. The observed resistance of females to Praziquantel seemed to depend on active proteins in the human serum. Moreover, different expression profiles of tegument-specific genes indicate different functions of female_single and male_single teguments in response to human serum. Our results indicate that female schistosomes developed different evasion strategies toward the host's immune system in comparison to males that might lead to more robustness and has to be taken into account for the development of new anti-schistosomal drugs.

Structure-function analysis of apical membrane-associated molecules of the tegument of schistosome parasites of humans: prospects for identification of novel targets for parasite control.

Neglected tropical diseases are a group of some 17 diseases that afflict poor and predominantly rural people in developing nations. One significant disease that contributes to substantial morbidity in endemic areas is schistosomiasis, caused by infection with one of five species of blood fluke belonging to the trematode genus Schistosoma. Although there is one drug available for treatment of affected individuals in clinics, or for mass administration in endemic regions, there is a need for new therapies. A prominent target organ of schistosomes, either for drug or vaccine development, is the peculiar epithelial syncytium that forms the body wall (tegument) of this parasite. This dynamic layer is maintained and organized by concerted activity of a range of proteins, among which are the abundant tegumentary annexins. In this review, we will outline advances in structure-function analyses of these annexins, as a means to understanding tegument cell biology in host-parasite interaction and their potential exploitation as targets for anti-schistosomiasis therapies.

Cercariae developing in Lymnaea natalensis Krauss, 1848 collected in the vicinity of Pretoria, Gauteng Province, South Africa.

Freshwater snails are known to serve as first intermediate hosts for various parasitic diseases such as schistosomosis and fasciolosis. Snails were collected on several occasions in the proximity of Pretoria, South Africa and their cercarial sheddings were studied. This article describes three different types of cercariae shed by the freshwater snail, Lymnaea natalensis, viz. a fork-tailed cercaria of a Trichobilharzia sp., an avian parasite belonging to the family Schistosomatidae, an echinostomatid cercaria of the family Echinostomatidae, also avian parasites and a xiphidiocercaria of the family Plagiorchiidae which parasitise avians and amphibians. The morphology of these cercariae was studied by light and scanning electron microscopy.

Schistosome monogamy: who, how, and why?

Schistosomes represent a unique animal model for comparative analyses of monogamy. Indeed, schistosomes are classified at the lowest taxonomical level of monogamous species and lack complex social interactions, which could alter our understanding of their unusual mating system. Elements discussed here include the fact that monogamy in schistosomes could be an ancestral state between hermaphroditism and polygyny or polygynandry and the occurrence of mate changes. In addition, hypotheses are proposed to explain monogamy in schistosomes (e.g. female dispersion, the need for paternal care, oviposition site limitation or aggressiveness, and mate guarding). We also propose future experimental and analytical approaches to improve our understanding of the schistosomes' mating system.

A proteomic approach to the identification of tegumental proteins of male and female Schistosoma bovis worms.

Schistosoma bovis, a parasite of ruminants, can live for years in the bloodstream in spite of the immune response of its host. The parasite tegument covers the entire surface of the worm and plays a key role in the host-parasite relationship. The parasite molecules involved in host immune response evasion mechanisms must be expressed on the tegument surface and are potential targets for immune or drug intervention. The purpose of the present work was to identify the tegumental proteomes of male and female S. bovis worms, in particular the proteins expressed on the outermost layers of the tegument structure. Adult worms of each sex were treated separately with trypsin in order to digest their tegumental proteins, after which the peptides released were analysed by LC-MS/MS for identification. This experimental approach afforded valuable information about the protein composition of the tegument of adult S. bovis worms. A range of tegumental proteins was identified, most of which had not been identified previously in this species. Although an absolute purification of the proteins expressed on the outermost layers of the tegument structure was not achieved, it is likely that present among the proteins identified are some of the molecules most closely associated with the tegument surface. Our study also suggests that there may be differences in the protein composition of the tegument of male and female schistosomes. Finally, the presence of actin and GAPDH on the surface of male and female worms and the presence of enolase exclusively on the surface of male worms were verified by confocal microscopy.

Towards tissue specific transcriptomics and expression pattern analysis in schistosomes using laser microdissection microscopy.

One difficulty facing post-genomic analyses of schistosomes is the limited data on sites of expression of many gene products expressed by the parasites in their hosts. The potential for use of laser microdissection microscopy as a preparative technique for transcriptional and proteomic profiling is reviewed. This technique allows tissues to be dissected for subsequent molecular and protein analysis. The method is reviewed in the light of the acoelomate triploblastic nature of tissue organisation in the parasite.

Schistosoma mekongi: the in vitro effect of praziquantel and artesunate on the adult fluke.

The efficacy and tolerance of 80 microg/ml praziquantel (PZQ) and 40 microg/ml artesunate (ATS) against adult stage Schistosoma mekongi in vitro were investigated after 3, 6, 12, and 24h incubation by monitoring worm motility and compared tegumental changes using scanning electron microscopy (SEM). Thirty mice were infected with S. mekongi cercaria for 49 days. Adult worms were collected by perfusion method and prepared for in vitro study. Contraction and decreased motor activity were observed after as little as 3h incubation with PZQ and ATS. Some of the worms were immobile 12h after exposure, and died within 24h. The tegument of S. mekongi showed severe swelling, vacuolization and disruption, fusion of the tegumental ridges, collapse and peeling. After 12-24h incubation, PZQ induced similar but they less severe, tegumental changes to those observed after exposure to ATS. The direct observation of the fluke motility and SEM study suggest that ATS is more effective than PZQ in causing tegumental damage in adult S. mekongi, and provides a basis for subsequent clinical trials.

Effects of praziquantel and artesunate on the tegument of adult Schistosoma mekongi harboured in mice.

The effects of praziquantel and artesunate on the tegument of adult Schistosoma mekongi harboured in mice were compared using scanning electron microscopy (SEM). Forty-two mice infected with S. mekongi for 49 days were treated intragastrically with either 300 mg/kg praziquantel or 300 mg/kg artesunate. Mice were sacrificed 1 or 3 days post-treatment. Worms were collected by perfusion and examined by SEM. One to 3 days after administration of artesunate, the tegument of S. mekongi showed severe swelling, vacuolization, fusion of the tegumental ridges and loss or shortening of the spines on the trabeculae, collapse and peeling. Praziquantel induced similar tegumental alterations as those observed after administration of artesunate, but they were less severe. Three days post-treatment, there was evidence of recovery only in the case of praziquantel. The results of our study suggest that artesunate is more effective than praziquantel in causing tegumental damage in adult S. mekongi, and provides a basis for subsequent clinical trials.

The involvement of cyclic adenosine monophosphate in the control of schistosome miracidium cilia.

This study examined the possible involvement of cyclic adenosine monophosphate (cAMP) in the control of ciliary action of Schistosoma mansoni miracidia. Miracidia immobilized in hypertonic NaCl solution were treated with 3 compounds that are known to increase intracellular cAMP concentrations. Forskolin, at a concentration of 50 microM, induced 50.1% of the miracidia to swim in hypertonic solution. The corresponding values obtained for 3-isobutyl-1-methylxanthine (IBMX) at 1 mM and 8-bromo-cAMP at 10 mM were 42.2 and 50.4%, respectively. The motility-enhancing effect of these compounds was dose dependent. Nevertheless, the swimming speed of miracidia activated in this way was only 10% of that observed in artificial pond water (APW). Cholera toxin had no apparent effect on miracidia swimming in hypertonic NaCl solution. Likewise, swimming in APW treated with forskolin at 50 microM, IBMX at 1 mM, or 8-bromo-cAMP at 10 mM did not induce any apparent change in motility. Miracidia swimming in APW were then treated with 3 compounds that decrease the intracellular concentration of cAMP. MDL-12,330A, at a concentration of 250 microM, caused a dramatic decrease in swimming over a period of 1 hr. Likewise, SQ22536 and imidazole, at concentrations of 20 and 50 mM, respectively, caused 36.5 and 73.4% decreases in swimming under the same conditions. Finally, inhibitors of cAMP-dependent protein kinase, i.e., PKI(14-22)amide, H89, and H88, completely inhibited miracidia swimming in APW at concentrations of 25, 50, and 100 microM, respectively. These results suggest that cAMP and cAMP-dependent protein kinase are involved in osmosis-controlled ciliary motion of schistosome miracidia.

Genomics of parasitic flatworms.

Although we live in what is often touted as the 'post-genomic era', this term is hardly appropriate when we consider the paucity of knowledge of the genomic biology of parasitic flatworms. The situation is, however, changing-at least for two species of Schistosoma. Recent transcriptome analysis of Schistosoma mansoni and of Schistosoma japonicum has identified novel genes and genes not previously reported for schistosomes, as well as the identification of the molecular mechanisms for host-dependent maturation, immune evasion, development, signalling and sexual dimorphism. The analyses also identify potential vaccine candidates and drug targets. Here, the current state of knowledge is reviewed for mitochondrial and nuclear genomes of parasitic flatworms. We highlight the remarkable recent progress in gene discovery for schistosomes and the goal of sequencing complete schistosome genomes.

Schistosoma ovuncatum n. sp. (Digenea: Schistosomatidae) from northwest Thailand and the historical biogeography of Southeast Asian Schistosoma Weinland, 1858.

Schistosoma sinensium Bao, 1958 was first isolated from an unidentified snail in Sichuan Province, PR China. This species was apparently rediscovered in Chiang Mai Province, northwest Thailand (Baidikul et al., 1984); the definitive host was the rat Rattus rattus and the intermediate host was the snail Tricula bollingi. In this paper S. sinensium is rediscovered in Sichuan Province and compared with worms recovered from experimentally infected mice, which had been exposed to cercariae shed by T. bollingi from Chiang Mai. Evidence is presented suggesting that the schistosome collected by Baidikul was not S. sinensium and that a new species is involved. The new species, named Schistosoma ovuncatum (etymology: ovum (egg) + uncatus (hooked)), is described and compared with related taxa. All previous papers on the Thai schistosome have used worms recovered from field-collected rodents only; this is the first account in which the life-cycle has been completed in the laboratory, using cercariae shed by T. bollingi, and the resulting worms described. S. ovuncatum differs from S. sinensium in terms of size and shape of body and egg, number of testes, size of ovary, length of vitellarium, intermediate host and biogeographical distribution. The relationships of the two taxa and their position with respect to the Schistosoma indicum- and S. japonicum-groups are discussed. The implications of the findings for the evolution of human schistosomiasis in the region are also commented upon.

Seeking the ghost of worms past.

The mechanisms of protective immunity to parasite infections in humans are still elusive. Here, Woolhouse and Hagan discuss new evidence suggesting that the extremely slow development of acquired immunity to human schistosomes may depend on exposure to antigens from these worms after they die.

Cercaria-schistosomulum surface transformation of Trichobilharzia szidati and its putative immunological impact.

Schistosome cercariae of the genus Trichobilharzia are the causative agent of swimmers' itch. In order to characterize the changes in parasites during and after the penetration of the host skin, in vitro and in vivo (in ducks and mice) transformations of T. szidati cercariae to schistosomula were performed. Ultrastructural observation revealed that cercariae possess a simple outer tegumental membrane with a thick glycocalyx. As with human schistosomes, the latter structure disappears during transformation and a new double membrane with putative protective function is formed. Our biochemical and immunological observations showed that the carbohydrate-rich glycocalyx of cercariae is readily bound by lectins and antibodies. The in vitro transformation to schistosomula can be detected by enhanced reactivity of 2 lectin probes (PNA and ConA) with the surface. The in vivo-transformed (skin and lung) schistosomula appear to have few surface ligands for the 12 lectin probes being tested. Similarly, the cercarial surface and its remnants on the in vitro-produced schistosomula is recognized by sera from immunized mice and humans with cercarial dermatitis; the tissue schistosomula fail to react with these antibodies. The loss of surface targets as a part of parasite immune evasion within the host is discussed.

Nutritional adaptations to parasitism within the platyhelminthes.

Some of the most significant alterations to the basic turbellarian plan are evident in the adaptations that relate to the acquisition of food by parasitic flatworms, reflecting the most potent of selection pressures in initiating and maintaining the host-parasite association. Nutritionally, ectoparasitic monogeneans show most correspondence with the predatory turbellarians, with certain monopisthocotylean members feeding by means of a protrusible pharynx and extracorporeal digestion, as skin-browsers of fish, with extensive intracellular digestion involving lysosomal enzymes in a well-differentiated gut. The more sheltered vascularised gill chamber of fish provides many polyopisthocotylean monogeneans with a totally renewable and more comprehensive diet in the form of blood, but haematophagy has necessitated a number of digestive adaptations, not least in resolving the problem of intracellular accumulations of haematin pigment. Haematophagy is the predominant feeding strategy of digeneans, but in contrast to monogeneans digestion of blood is largely extracellular; in schistosomes digestion is rapid, involving a battery of cathepsin-like cysteine proteinases and aminopeptidases. The external surfaces of all parasitic flatworms depart from turbellarian character and are composed of a multifunctional syncytial tegument, which is permeable to a variety of small organic solutes, some crossing by passive diffusion, others via facilitated or active mediated transport. The relative roles of the tegument and gut in trematode nutrition are difficult to assess, but can be related to the nature of the microhabitat within the host. Cestodes are highly adapted intestinal parasites bereft of any vestige of gut, and their tegument has become elaborated into a sophisticated and highly efficient digestive-absorptive layer, rivalling the vertebrate mucosa in its ability to gain kinetic advantage in the selective uptake of nutrient at the host-parasite interface. The patterns of energy metabolism in adult flatworm parasites are generally anaerobic and based on glycogen, with abbreviated metabolic pathways and the loss of biosynthetic capacities.

Variations in Schistosoma mattheei egg morphology and viability according to age of infection in cattle.

Comparison of the numbers of Schistosoma mattheei eggs and miracidia per gram faeces in groups of naturally infected calves, heifers and adult cows showed that the reduction in faecal egg excretion recorded as infection progresses is associated with a decline in the ability of eggs to hatch. While 50% of the eggs from calves produced a miracidium, only 15% of those excreted from adult cows did the same. The decline in egg viability is at least partly associated with morphological changes of the eggs. About twice as many smaller and vacuolated eggs were found in the faeces of heifers and adult cows (33.8%) as compared to animals in early infection (16.1%).

Morphometric variability of Schistosoma intercalatum eggs: a diagnostic dilemma.

Variability of Schistosoma intercalatum eggs in shape and size, and their similarity to those of S. haematobium presented a problem of species identification when egg morphology was the diagnostic criterion used in a study of human schistosomiasis conducted on São Tomé and Principe. More than 2500 egg measurements were obtained by light micoscopy to gather data relating to size variability of S. intercalatum eggs, to evaluate whether factors such as age of host, sex of host and intensity of infection are correlated with variability, and the data were compared with previously published measurements on different isolates and strains of S. intercalatum: the range in length (104-203 microns) embraces most of the measurements reported in other studies of S. intercalatum eggs. There was no correlation either between age and sex of the host, or intensity of infection with variability of egg size. Comparison between measurements of the eggs of S. haematobium, S. intercalatum and S. bovis eggs are presented.

A simple computer-assisted method to identify schistosome cercariae.

Several methods have been suggested to identify schistosome cercariae. In the present work, a new method is proposed, based on the analysis of the distribution of sensory endings (sensillae) on the body of the cercariae, revealed by an impregnation with silver nitrate. We determined the mutual distances between the sensillae and calculated the mean values, standard deviations, coefficients of asymmetry, and of kurtosis of the distribution of these mutual distances. Applied to two species, Schistosoma mansoni from Brazil and S. intercalatum from Cameroon, these mutual distances had the same mean value and the same standard deviation but quite different coefficients of symmetry (0.34 +/- 0.11 versus 0.73 +/- 0.08; P < 0.0001) and of kurtosis (-0.82 +/- 0.27 versus -0.58 +/- 0.31; P < 0.0001). The latter two indices were therefore very effective for discriminating the two species. The present method can be applied to other species and to hybrids in the field.

ITS2 ribosomal RNA indicates Schistosoma hippopotami is a distinct species.

The internal transcribed spacer region of the ribosomal RNA, ITS2, was sequenced from a single specimen of S. hippopotami collected from a pulmonary artery of the hippopotamus, Hippopotamus amphibius in South Africa. The nucleotide sequence was aligned with those of S. mansoni, S. rodhaini, S. haematobium, S. intercalatum, S. curassoni, S. bovis and S. japonicum. Both maximum parsimony and genetic distance analyses were performed on these data sets. Using S. japonicum as outgroup to the African schistosomes, a single most-parasmonious tree was obtained of length 64 steps with a consistency index of 1-S. hippopotami was the sister-group to the remaining African species. This species has lateral-spined eggs and its basal position in the tree suggests that this condition is primitive and that terminal-spined eggs developed secondarily. Molecular data clearly show that S. hippopotami cannot be considered synonymous with S. mansoni. Assuming the hippopotamus is the normal host of S. hippopotami, phylogenetic analysis is consistent with an ancient association between schistosomes and ungulates.

Schistosomiasis in the Republic of São Tomé and Principe: characterization of Schistosoma intercalatum.

This paper reports the morphological and biochemical characterization of the species of Schistosoma infecting humans in the Republic of São Tomé and Principe. The eggs are typical in shape and size of S. intercalatum, measuring on average between 174.5 microns and 189.1 microns. The eggs are voided in the faeces and not the urine of infected people. The parasite experimentally develops in several different species of Bulinus belonging to the B. forskalii group, including B. forskalii, with a minimum prepatent period of 25 d, and also in snails of the B. reticulatus group (B. wrighti); it is incompatible with snails of the B. africanus and B. truncatus/B. tropicus complex. A survey of 5 different habitats at intervals of 2 weeks over a period of one year showed that populations of B. forskalii increased during the dry period of June, July and August in 1988, and in 3 of the habitats snails were present throughout the year. Hence transmission may take place in these habitats throughout the year. Preliminary evidence suggests that water velocity is a limiting factor confining Bulinus to the north-east of the island where the terrain is less mountainous. Development of schistosomes from São Tomé was followed in experimentally infected hamsters. The cross-over point (the point at which the paired male and female worms are of the same average length) occurred at about 49 d after infection: eggs were first seen in the uteri of the female worms 48 d after infection. The parasite from São Tomé developed in sheep and produced viable eggs.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro transformation of the miracidium to the mother sporocyst of Schistosoma margrebowiei; changes in the parasite surface and implications for interactions with snail plasma factors.

The in vitro transformation of the miracidium to the mother sporocyst of Schistosoma margrebowiei was initiated by placing the miracidium in mammalian physiological saline. The transformation occurs in stages: the cilia cease beating; the ciliated plates become detached from the intercellular ridges and underlying muscle layers; the intercellular ridges spread over the body surface eventually forming a new tegument; the sporocyst changes from an ovoid to a tubular shape in about 48 h at room temperature. The surfaces of the miracidium, sporocyst and cercaria of S. margrebowiei display stage-specific carbohydrates on their surfaces as indicated by lectin staining. Ricin120 stains the cilia alone of the miracidium whereas peanut agglutinin stains the larval surface except for the cilia. The intercellular ridges of the miracidium stain with concanavalin A and wheat germ agglutinin, and these lectins stain the entire surface of the mature mother sporocyst. The cercaria is the only larval stage which stains positively with asparagus pea lectin. Bulinus nasutus is incompatible with Schistosoma margrebowiei; the haemolymph of this snail contains an agglutinin which agglutinates a wide variety of mammalian erythrocytes including those of human ABO blood groups. The haemagglutinin titre of B. nasutus plasma is reduced after incubation with miracidia of S. margrebowiei indicating that the agglutinin is absorbed onto the surface of this larval stage but not that of the mother sporocyst or cercaria. The possible roles of agglutinins in host-parasite interactions together with the significance of the differences in the surface carbohydrates of the larval stages are discussed.