We investigated the involvement of nitric oxide in Schistosoma-induced liver injury. We found that inducible nitric oxide synthase mRNA became detectable in the liver at the onset of parasite egg laying and levels then increased as the eggs accumulated in the organ. Enzyme concentration and activity paralleled mRNA levels. The event was a direct effect of egg deposition, as it occurred in the liver after natural infection, or in the lungs after i.v. injection of the eggs. However, nitric oxide seems to have no direct effect on the eggs since in vitro assays showed that the nitric oxide donor SIN-1 did not alter the ability of the eggs to hatch. L-Arginine and L-NAME, a nitric oxide synthase inhibitor, were administered to infected mice in an attempt to increase or reduce nitric oxide production, respectively. Arginine had no effect on the disease, whereas the inhibitor led to a marked decrease of hepatic injury with, in particular, reduced fibrosis and decreased lipid peroxidation. In conclusion, not only is inducible nitric oxide synthase activity unlikely to exert an anti-microbicidal effect against the egg stage of S. mansoni but it might lead to deleterious effects in the liver and therefore contribute to the pathology.

Immunological data indicate that different subsets of T-helper cells work best against different types of infection. Concomitant infection of a host may thus impose either conflicting or synergistic immune response requirements, depending upon the extent to which the component optimal immune responses differ. Drawing upon empirically-determined optimal responses to single-species infections, an optimality model is here used to generate testable hypotheses for optimal responses to concomitant infection. The model is based upon the principle that the joint immune response will minimize divergence from each of the optima for single-species infections, but that it will also be weighted by the importance of mounting the correct response against each infectious organism. The model thus predicts a weighted average response as the optimal response to concomitant infection. Data on concomitant infection of murine hosts by the parasites Schistosoma mansoni and Toxoplasma gondii will provide the first test of the optimality model. If the weighted average hypothesis holds true, then there are no emergent immunological properties of concomitant infections and we may be able to understand immune responses to concomitant infection directly via our understanding of single-species infections.

There is evidence that faecal egg counts of Schistosoma mansoni vary considerably from day to day, which results in poor sensitivity of single stool readings. Intra-specimen variation of S. mansoni egg counts may also be considerable, but has previously been considered as the less important component. We quantified the relative contribution of these two sources of variation among 96 schoolchildren from an area in Côte d'Ivoire highly endemic for S. mansoni. Stool specimens were collected over 5 consecutive days, and 5 egg-counts were made in each specimen by the Kato–Katz technique. The point prevalence of the first sample was 42.7% and the cumulative prevalence after the maximum sampling effort was 88.5%. Using generalized linear mixed models we found that the presence of S. mansoni eggs in a stool sample varied much more between days than within specimens, indicating that stool sample examination over multiple days is required for accurate prevalence estimates. However, using the same approach, we found that among infected children intra-specimen variation in egg counts was 4.3 times higher than day-to-day variation. After praziquantel administration, day-to-day variation was more important than before, since most infections were very light and thus likely to be missed altogether by stool examination on a single day. We conclude that diagnostic sensitivity in high transmission areas is maximized by making several stool readings on several days, but examining 1 stool specimen several times can make reasonable estimates of infection intensity.

The aggressive use of praziquantel to combat schistosomiasis in Egpyt raises concern about the possible emergence of resistance. Eggs from Egyptian patients with praziquantel-resistant infections (not cured by 3 doses of praziquantel) have been used to establish infection-specific schistosome isolates in mice. The response of these worms to the drug was observed in vitro, in order to determine if the isolates obtained from these resistant infections were, in fact, less responsive to praziquantel. One of the hallmark effects of praziquantel on schistosomes in vitro is a disruption of the worm's outer surface, the tegument. Here, praziquantel-induced tegumental damage is observed in 3 distinct isolates, 2 derived from resistant infections and 1 from an infection cured by a single dose. The isolates from the resistant infections were less susceptible to praziquantel-induced tegumental damage in vitro, suggesting that the worms are in some way less responsive to the drug.

Growth and development of adult schistosomes requires permanent communication processes of the parasites with their specific host environment and, additionally, between the two genders. Accumulating evidence suggests that, at the molecular level, the mandatory interactions are mediated by signal transduction processes. During recent years, a considerable interest has emerged in the identification of signalling molecules from this parasite and to elucidate their roles during development. In this organism, a number of different molecules have been identified which belong to diverse classes of evolutionary conserved signal transduction cascades. However, up to now no representative of the conserved family of cellular tyrosine kinases has been identified. In this study we present a suitable approach to identify this class of molecules and demonstrate the successful cloning and molecular characterization of one of the isolated genes, the tyrosine kinase 5 (TK5). An unexpected finding was that in the Liberian strain of Schistosoma mansoni the TK5 gene exhibits an allelic polymorphism.

Haematophagous insect vectors of arthropod-borne viruses (arboviruses) feed repeatedly. Consequently, they can transmit arboviruses to more than one host during the same developmental stage (intra-stadial transmission). By contrast, ixodid ticks generally feed only once at each parasitic stage (larva, nymph, and adult) and hence they have only one opportunity for tick-borne virus transmission per stadium (inter-stadial transmission). Under natural conditions, tick-infested hosts may die (from disease or other causes) before the ticks have completed their long period of engorgement. A laboratory model was used to investigate the consequences of premature host death on tick-borne virus transmission. We report intra-stadial transmission of Thogoto virus by the nymphal, male, and female ticks of Rhipicephalus appendiculatus. Tick-borne Thogoto virus infection caused viraemia and death of hamsters before the nymphal and adult ticks feeding on them had completed engorgement. The resulting partially fed ticks were allowed to continue engorgement on new, uninfected hosts (interrupted feeding). During feeding on the subsequent hosts, they transmitted the virus intra-stadially to susceptible hosts (hamsters), and to uninfected co-feeding ticks on non-susceptible hosts (guinea-pigs). Intra-stadial transmission, mediated by interrupted feeding, may help explain outbreaks of rapid and fatal tick-borne viral diseases, and the epidemiology as well as evolution of virulence, in a susceptible host population. Additionally, intra-stadial transmission provides an increased risk of tick-borne pathogen transmission to humans and domestic animals during slaughter and game hunting.

More than 20 species of the entomopathogenic nematode Steinernema have been described; to date, all reproduce exclusively by cross-fertilization of male and female individuals. Steinernema sp. strain T87 from Indonesia was found to consist largely of self-fertile hermaphrodites. Progeny were produced by morphological females both in insects (Galleria mellonella) and in hanging drops of insect haemolymph inoculated with a single infective juvenile. Sperm were present in the oviduct of unmated morphological females. Approximately 1% of infective juveniles developed into males, and males were also present in the second generation where they constituted 1–6% of the population. Under the same conditions the related species Steinernema longicaudum strain CB2B displayed typical steinernematid reproduction: cross-fertilization and a 1:1 sex ratio. It is argued that the development of hermaphroditism in Steinernema sp. T87 represents convergent evolution with Heterorhabditis, the other major genus of entomopathogenic nematode.

In the last two decades, major immunodeficiency syndromes have strongly influenced medical parasitology. Some animal parasitoses, once unknown in human medicine, have become zoonotic and sometimes anthroponotic. In other cases, the clinical evolution of human parasitoses has been severely aggravated and/or modified in immunodeficient patients especially in toxoplasmosis, cryptosporidiosis, leishmaniasis, strongyloidiasis and scabies. The parasites implicated are varied (protozoa, helminths and even Acaridae) but have in common the capacity to reproduce in or on the human host. These immunodeficiency syndromes are often related to AIDS but other major immunodepressions, such as post-therapeutically in organ transplantation, may also be responsible and raise difficult problems for prevention. The immunological mechanisms involved are not always well understood. In addition, genetic predisposition factors, gradually becoming better-understood in parasites and man, complete and complicate our understanding of the immunological mechanisms.

In contrast to the growing knowledge accumulated on plant resistance to pathogens, mechanisms of parasite resistance largely remain to be elucidated in animal species. In the present study we investigated mechanisms underlying resistance/susceptibility in the snail–trematode system Biomphalaria glabrata–Echinostoma caproni. In particular, we compared the effect of the parasite excretory–secretory (E–S) products on the defence functions of haemocytes from 2 susceptible and 2 resistant snail strains. In vitro experiments showed that E. caproni E–S products inhibit adhesion and phagocytosis of haemocytes from susceptible snails. A partial biochemical characterization also suggested that the interfering factor(s) is (are) heat-labile glycosylated polypeptides of molecular mass between 10 and 30 kDa. Interestingly, haemocytes from resistant snails remained unaffected by the parasite E–S products, suggesting that a constitutive difference results in their refractoriness to the parasite's immunosuppressive factor(s).

We have investigated the development of the schistosome egg and its secretions in order to understand how it migrates through gut tissues and also initiates pathology in the liver. We show by electron microscopy that the subshell envelope is absent in the newly deposited egg, but appears very early and differentiates as development progresses. In the mature egg, this nucleated envelope contains extensive endoplasmic reticulum, suggestive of a protein synthetic capacity. Furthermore, Reynolds' layer only appears between the envelope and the egg-shell in the mature egg and may represent its accumulated secretions. We have biosynthetically labelled and collected the secretions (ESP) released by mature but not immature eggs during culture. Their fractionation by SDS–PAGE reveals a simple pattern of 6 bands, differing markedly in composition from soluble egg antigen preparations. Electrophoresis in casein substrate gels demonstrates the presence of 2 distinct proteases in the egg secretions. By immunocytochemistry, ESP localized predominantly to the envelope of the mature egg, suggesting that this layer rather than the miracidium is the source of egg secretions.

The physiological effects of selected classical transmitters and FMRFamide-related peptides (FaRPs) on dispersed muscle fibres from the marine turbellarian, Procerodes littoralis have been examined. Confocal scanning laser microscopy coupled with fluorescein isothiocyanate (FITC) or tetramethylrhodamine (TRITC)-labelled phalloidin revealed a highly developed body wall muscle system with circular, longitudinal and diagonal layers of muscle fibres. Dispersed muscle fibres contracted when depolarized by exposure to extracellular media with elevated K+ (15–100 mM) in a concentration-dependent manner, with a maximal response of 87% achieved at [ges ] 75 mM. 5-Hydroxytryptamine (5-HT) induced concentration-dependent muscle contraction between 0·01 and 1000 μM, with 10 μM producing a near maximal contraction response of 75%. Acetylcholine (ACh) had less pronounced excitatory effects (0·01–1000 μM), inducing contraction of only 32% of the fibres at 100 μM. The flatworm FMRFamide-related peptides (FaRPs), GYIRFamide, YIRFamide and GNFFRFamide each had concentration-dependent myocontractile effects, indicating the occurrence of at least 1 FaRP receptor on P. littoralis muscle fibres. At 10 μM peptide, GNFFRFamide induced contractions in < 40% of the muscle fibres examined, whereas YIRFamide and GYIRFamide induced contraction in 70 and 75%of muscle fibres, respectively. The order of potency of the peptides was: GYIRFamide > YIRFamide > GNFFRFamide. Pre-incubation of the muscle fibres in 5 μM 5-HT significantly reduced the responses to GYIRFamide, YIRFamide and 5-HT, while the responses to high K+ remained unaltered. Muscle fibres pre-incubated in GYIRFamide (0·1 μM) were also less responsive to 5-HT but not to ACh and high-K+. The GYIRFamide analogue, GYIRdFamide, did not induce muscle contraction (0·01–100 μM) per se, but when co-applied with the myoactive peptides GYIRFamide, YIRFamide or GNFFRFamide, it significantly blocked their ability to elicit contractions. This suggests that the peptides tested may act via a common muscle-based neuropeptide receptor. GYIRdFamide did not alter the contractile effects of high K+, 5-HT or ACh. Collectively, these results indicate that FaRPs, 5-HT and ACh all have the potential to cause muscle contraction in flatworms and that 5-HT and FaRPs alter muscle sensitivity to each other, but do not influence the ability of flatworm muscle fibres to contract.

Parasitic worm infections are amongst the most widespread of all chronic human infections. It is estimated that there are more than 3 billion infections in the world today. In many low income countries it is often more common to be infected than not to be. Indeed, a child growing up in an endemic community can expect be infected soon after weaning, and to be infected and constantly reinfected for the rest of her or his life. Infection is most common amongst the poorest and most disadvantaged communities, and is typically most intense in children of school going age. As the risk of morbidity is directly related to intensity of infection, it follows that children are the most at risk from the morbid effects of disease. Multiparasite infections are also common in such communities and there is evidence that individuals harbouring such infections may suffer exacerbated morbidity, making children even more vulnerable. Thus, these infections pose a serious threat to the health and development of children in low income countries. For many years, the need to control these infections has lain uncontested, and with the advent of broad-spectrum anthelminthic drugs that are cheap, safe and simple to deliver, control has at last become a viable option for many communities. Furthermore, there is now increased emphasis being placed on a multispecies approach as a cost-effective mechanism to control the morbidity of virtually all the major helminthic infections of humans.

Random amplified polymorphic DNA (RAPD) markers were used to quantify genetic diversity within and between 5 populations of Schistosoma mansoni within its definitive host (Rattus rattus) and the 5 corresponding populations of the snail intermediate host (Biomphalaria glabrata) from a limited endemic area of murine schistosomiasis on the island of Guadeloupe. Analysis of molecular variance (AMOVA) and canonical correspondence analysis (CCA) were used to test the significance of genetic differentiation between populations. Both methods gave similar results. Of total gene diversity, 15.1% (AMOVA) and 18.8% (CCA) was partitioned between localities for S. mansoni with an absence of association between genetic and geographical distances. Geographical localities accounted for 20.5% (CCA) of the total diversity for B. glabrata populations. The genetic distances between pairs of parasite populations were not correlated with the genetic distances between the corresponding pairs of snail host populations. Such strong patterns of local differentiation of both parasite and snail populations are consistent with predictions based on metapopulation dynamics and may have implications on host–parasite susceptibility relationship through local adaptation processes.

If the deleterious effects of non-specific parasites are greater on vulnerable host species than on reservoir host species then exclusion of the vulnerable host through apparent competition is more likely. Evidence suggests that such a mechanism occurs in interactions between the ring-necked pheasant (Phasianus colchicus), the grey partridge (Perdix perdix), and their shared caecal nematode Heterakis gallinarum. Modelling of the system predicts that the reduced parasite impact on the pheasant compared to the partridge results in the force of infection transmitted from pheasants to partridges being sufficient to cause partridge exclusion. Since the parasite impacts are currently estimated from correlational work, controlled infections were conducted to experimentally compare the impact of H. gallinarum on the two hosts and verify cause and effect. While challenged partridges showed reduced mass gain, decreased food consumption, and impaired caecal activity, in comparison to controls, the only detectable effect of parasite challenge on the pheasant was impaired caecal activity. The impact of H. gallinarum on challenged partridges conforms with previous correlational data, supporting the prediction that parasite-mediated apparent competition with the ring-necked pheasant may result in grey partridge exclusion. However, the observed decrease in the caecal activity of challenged pheasants could imply that H. gallinarum may also have an impact on the fecundity and survival of pheasants in the wild, particularly if food is limiting. If this is the case, the associated decrease in the force of infection to which the partridge is exposed may be sufficient to change the model prediction from partridge exclusion to pheasant and partridge coexistence.

During trans-dermal invasion of the vertebrate host, larval schistosomes (cercariae) transform into schistosomula and become enveloped by a double lipid bilayered, tegumental membrane. The glucose transporter protein SGTP4 is found exclusively in these host-interactive tegumental membranes and in membranous vesicles proposed to be their precursor. In this study, we monitored the appearance and migration of this tegumental marker protein during larval transformation to test the current model of tegumental membrane biosynthesis in parasitic blood flukes. Only minutes after transformation was initiated, SGTP4 began accumulating in a previously unrecognized, bilaterally symmetrical, ‘cyton network’ beneath the peripheral muscle. Approximately 30min after the initiation of transformation the marker protein was seen in tubules connecting the network to the surface and erupting onto the surface in discrete patches. After 1h the patches were regularly arrayed over the schistosomula body and began to cover the anterior organ. By 3h the staining has largely resolved into a contiguous layer of fluorescence covering most of the worm surface. These findings confirm earlier suggestions, based on electron microscopy, that the parasite's surface tegumental membranes are derived from the migration of membranous vesicles produced within cytons and reveal a new subtegumental architecture interconnecting the cytons.

The toxicity of cadmium, zinc and cadmium/zinc mixtures at concentrations ranging from 1000 to 50000μg/l were investigated against cercariae and metacercariae of Parorchis acanthus obtained from the dog whelk Nucella lapillus. Cercarial encystment at concentrations of 25000μg/l or higher was significantly impaired by all test metals; however, at lower concentrations only zinc demonstrated toxicity. Mixtures of cadmium and zinc had a synergistic effect compared with single metal toxicity but only at 50000μg/l. Excystment in vitro was only significantly affected by cercariae exposed to cadmium/zinc mixtures whilst encysting. Twenty-four h exposures of fully formed cysts had no effect on excystment in vitro. Effects on in vitro excystment rates over a 2h period demonstrated widespread effects for cercariae-exposed P. acanthus. No effects were evident on excystment rates of cyst-exposed parasites.

Immunological and structural changes in the thymus and pronephros of Cyprinus carpio infected with the blood fluke, Sanguinicola inermis for 30 days, and exposed to 0·5mg NH+4/l for 48 and 168h were investigated. Ultrastructural observations revealed cell disruption and highly vacuolated cytoplasm in the thymus. Of the cells that remained intact there was a significant increase in thrombocytes after 48h exposure to the pollutant. In addition, there was a decrease in lymphocytes following exposure to ammonia at both time-periods studied. In contrast the pronephros of fish exposed to the pollutant underwent relatively mild changes in cellular architecture although ammonia and time of exposure had significant effects on the proportions of several leucocyte types. A significant decrease in neutrophils, thrombocytes and lymphocytes occurred in fish exposed to the pollutant for 168h. Pronephric lymphocyte stimulation (cpm) by Con A and PWM increased in vitro, whereas the stimulation index was reduced in infected fish exposed to ammonia. Changes in the immune organs of S. inermis-infected carp treated with pollutant were both organ- and time-specific. The possible reasons for this are discussed and significance in relationship to parasitization assessed.

This study investigated sequence variability in the second internal transcribed spacer of ribosomal DNA for 3 species of Metastrongylus (porcine lungworms). The ITS-2 region was amplified by PCR from individuals of M. elongatus, M. pudendotectus and M. salmi, and then subjected directly to single-strand conformation polymorphism analysis (SSCP), which allowed the direct display of sequence variation within and among individuals representing each speciesNucleotide sequences data reported in this paper are available in the EMBL, GenBankTM accession numbers: AJ305373 to AJ305404.. There were marked differences in SSCP profiles among species, making this approach useful for species identification. For individual species, representative bands were excised from electrophoretic gels, reamplified by PCR and subjected to direct sequencing. For all 3 taxa, variability in the ITS-2 was related chiefly to the presence of microsatellites. Eight different microsatellites were identified, namely (A)n, (TG)n, (TCG)n, (TA)n, (TATG)n, (G)n, (TACA)n and (T)n. Considerable variability in microsatellite repeat number (ranging from 1 to 23) was found among individual nematodes of a species and between species. The microsatellites were located to specific stem or loop regions in the predicted ITS-2 precursor rRNA secondary structure. The results may suggest that slipped-strand mispairing in microsatellite regions contributes to sequence variability in the ITS-2 of Metastrongylus species under structural constraint as a consequence of microsatellite location in the precursor rRNA. Similar studies of the ITS-2 for a wide range of parasitic nematodes may lead to a better understanding of concerted evolution in these organisms.

The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is no in vitro propagation system for S. scabiei available, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed an S. scabiei cDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. Recombinant S. scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed in E. coli and was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of important S. scabiei antigens and that recombinant proteins can be useful for the study of scabies.