Intestinal helminth parasites in flounder Platichthys flesus from the River Thames: an infracommunity analysis.

An analysis was undertaken of intestinal helminth communities in flounder Platichthys flesus from two sites on the River Thames. A comparison was made between helminth community richness and diversity from these sites at the component and infracommunity levels. At the component community level, a richer and more diverse parasite community was found in flounder from the Tilbury location (marine influence) than that from the Lots Road location (freshwater influence). At the infracommunity level, more parasite species and parasite individuals per host were found at Lots Road and the percentage of similarity values were low at both locations. Helminth species with high prevalence values in the parasite communities of the flounder are the dominant species in any individual fish, harbouring multi-specific infections. The presence of more invertebrate species, which are intermediate hosts in the helminth life cycle in the Thames, fish vagility and the high prevalence and abundance values of Pomphorhynchus laevis in the flounder, may explain the differences between the two locations.

A comparison of topical formulations for the prevention of human schistosomiasis.

Recently, a dimeticone formulation has been shown to be effective at preventing Schistosoma cercariae infecting skin, while DEET (N,N-diethyl-m-toluamide), a highly effective insecticide, has been shown to have activity against cercariae. Seven formulations, 3 containing DEET, were prepared and applied to excised human skin in Franz cells for 1 h. Schistosoma cercariae were applied for 30 min at 1 and 24 h, and the number that penetrated the skin calculated (n = 9). DEET could not be incorporated into the dimeticone formulation, yet it remained the most effective at preventing cercarial penetration, both 1 and 24 h after application. The ointments that contained DEET did prevent penetration but their mode of action was due to the toxicity of DEET against the cercariae. The persistence of the protection afforded by the dimeticone formulation after washing suggests that the formulation may be interacting with the stratum corneum to prevent cercarial recognition of skin.

Pallisentis rexus from the Chiang Mai Basin, Thailand: ultrastructural studies on egg envelope development and the mechanism of egg expansion.

Pallisentis rexus Wongkham & Whitfield, 1999 (Eoacanthocephala: Quadrigyridae) infects the freshwater snakehead fish, Channa striata, in the Chiang Mai Basin, Thailand. All stages of egg development within the body cavity of the female parasite were observed, using transmission electron microscopy. Changes in mature eggs after contact with water were also investigated. The mature egg has five egg envelopes separated from each other by four gaps. The fertilization membrane, which is formed first, is pushed centrifugally by other, subsequently formed, envelopes and gaps, which produces a final total shell thickness of 8-36 microm around the acanthor. The disappearance of the outermost layer and the unpleating of an adjacent inner layer causes the expansion of eggs on contact with water. The volume of an expanded egg is approximately 27 times that of an unexpanded one, but the density of eggs is reduced from a value greater than water to one almost equal to water. This is believed to aid the dispersion of eggs.

Age-dependent survival and infectivity of Schistosoma mansoni cercariae.

The age dependency of the mortality, spontaneous de-tailing and infectivity of cercariae of Schistosoma mansoni has been determined at 25 degrees C. Infectivity was assessed with respect to stratum corneum-like differentiated human keratinocyte cultures (validated by comparison with fresh human skin samples) and displayed a complex age-dependent pattern. From 1 to 9 h post-emergence cercariae showed a plateau of maximal infectivity (around 90% attachment). Thereafter, infectivity declined. Immediately after release, infectivity at around 60% was significantly lower than the plateau values and this could be an adaptation for spatial dispersal of cercariae. Age-dependent patterns of cercarial mortality and spontaneous de-tailing closely mirrored the infectivity pattern except in relation to the low initial infectivity value. These findings suggest that, at a population level, the age-dependent decline in cercarial infectivity towards human skin is essentially driven by cercarial mortality. The recently described phenomenon of delayed tail loss (DTL) in S. mansoni cercariae infecting human skin is confirmed in the present study. For cercariae aged up to 13.5 h post-emergence, 90% or more of invading cercariae took their tails with them into the keratinocyte culture. The infection dynamics described in this study suggest that diurnally shed S. mansoni cercariae, with peak emergence around mid-day, will have near maximal infectivity towards humans in contact with water through all remaining daylight hours in the tropics.

Delayed tail loss during the invasion of human skin by schistosome cercariae.

Schistosomiasis is initiated when cercarial larvae invade human skin. Contrary to long-held assumptions, most cercariae of Schistosoma mansoni do not shed their propulsive tails as they penetrate. Scanning electron microscopy studies and infection experiments with entire human skin and differentiated, stratum corneum-like, human keratinocyte cultures, have shown that most cercarial tails enter the skin along with their bodies. We propose that this behaviour is an adaptive trait linked with concomitant immunity.

Penetration of human skin by the cercariae of Schistosoma mansoni: an investigation of the effect of multiple cercarial applications.

It has previously been postulated that L-arginine emitted by penetrating Schistosoma mansoni cercariae serves as an intraspecific signal guiding other cercariae to the penetration site. It was suggested that penetrating in groups offers a selective advantage. If this hypothesis is correct and group penetration at one site on the host offers an advantage, it would follow that at such a site, successive groups of cercariae would be able to penetrate skin in either greater numbers or at a faster rate. This prediction was tested by the use of an in vitro model of cercarial penetration based on the Franz cell and using human skin. It was demonstrated that there was no increase in the percentage of cercariae able to penetrate the skin with subsequent exposures. Consequently, it seems unlikely that the release of L-arginine by cercariae during penetration could have evolved as a specific orientation system based on a selective advantage offered by group penetration.

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.

Antischistosomal bioactivity of isoflavonoids from Millettia thonningii (Leguminosae).

Extracts of the seeds of the leguminous tree Millettia thonningii are molluscicidal and schistosomicidal and contain a mixture of isoflavonoids and 3-phenylcoumarins. Robustic acid and a pure mixture of alpinumisoflavone and dimethylalpinumisoflavone, as characterized by nuclear magnetic resonance, were isolated from a dichloromethane extract. The bioactivity of these compounds against Schistosoma mansoni miracidia, cercariae, and adult worms was tested in vitro. Both robustic acid and the alpinumisoflavone mixture killed cercariae and adult schistosomes in vitro. At sublethal concentrations of the isoflavonoids, reductions in miracidial motility were measured using a video-imaging technique. The inhibition of mitochondrial electron transport at site I by the isolated isoflavonoids was also investigated. The results suggest that robustic acid and at least 1 alpinumisoflavone compound from the dichloromethane extract of the seeds of M. thonniogii are responsible for some of the observed bioactivity of this extract against schistosomes.

The attachment of Schistosoma mansoni cercariae to human skin cells.

Most of our knowledge about the process of penetration of skin, by cercariae of Schistosoma mansoni, has been gained from studies carried out in vivo with laboratory animals. Human skin is significantly different from that of other animals but there are obvious practical difficulties in directly studying attachment and penetration with human skin. Techniques have been developed which enable a 3-dimensional skin equivalent to be grown in tissue culture, made from different types of human skin cells. The aim of the present study was to investigate cercarial interactions with confluent cultures of the individual skin cell types that make up normal human skin and which will be used to construct a multi-component model. Cercariae behaved differently towards the various cell types tested. They responded least to monolayers of endothelial cells and most to primary keratinocytes, derived from human foreskin and differentiated at an air/liquid interface. This study demonstrates, therefore, that cercariae are capable of distinguishing between different types of skin cells and they preferentially attach to differentiated cells which form the epidermis.

A comparison of freshwater and marine/estuarine strains of Pomphorhynchus laevis occurring sympatrically in flounder, Platichthys flesus, in the tidal Thames.

Collections of flounder, Platichthys flesus, at two sites on the tidal River Thames in 1994 and 1995 have, for the first time, revealed the sympatric occurrence of the freshwater and marine/estuarine strains of the acanthocephalan parasite Pomphorhynchus laevis. This natural co-occurrence of the strains has been employed to compare infection levels and a range of parasite attributes of the two strains under conditions of sympatry. At both Lots Road (Chelsea) and Tilbury the marine/estuarine strain was present at far higher infection levels than the freshwater form. In a detailed comparison of worms from Tilbury flounder, a range of differences was revealed between the two strains. In single strain infections in individual fish, freshwater and marine/estuarine worms had distinct but overlapping gut microhabitat use patterns, with the former having a central intestinal bias and the latter a bias for the posterior region of the gut. In mixed strain infections, niche contraction occurred so that no overlap occurred. Freshwater worms were larger and had more eggs, more ovarian balls, and a higher percentage of fully developed eggs than the marine/estuarine worms. These differences are thought to reflect intrinsic, presumably genetically determined, differences between the two strains as they occurred in the same fish host species collected at the same place and time. Apparent differences in strain reproductive potential in flounder in the tidal Thames are discussed in the context of previous studies and the intermediate host segment of the parasite life cycle.

Inhibition of Site I mitochondrial electron transport by an extract of the seeds of Millettia thonningii: a potential mechanism for the plant's molluscicidal and schistosome larvicidal activity.

A dichloromethane extract of the seeds of Millettia thonningii (Leguminosae) which contains a mixture of isoflavonoids (predominantely robustic acid, alpinumisoflavone and dimethylalpinumisoflavone) is known to have larvicidal activity towards the miracidia and cercariae of schistosomes and to possess significant molluscicidal activity. The present investigation has assayed the effects of this extract on the electron transport systems of isolated rat liver mitochondria. The extract was found to inhibit mitochondrial electron transport at Site I (NADH dehydrogenase) at concentrations of 30-159 mg x l(-1). Although the extract is not as potent an inhibitor at Site I as rotenone, a known inhibitor of NADH dehydrogenase, such observations could explain the molluscicidal and schistosomicidal activity of dichloromethane extracts of the seeds of M. thonningii.

The infection of human skin by schistosome cercariae: studies using Franz cells.

Franz cells (2-chambered, air/fluid phase static diffusion devices, previously used for the study of drugs across viable human skin) are utilized for the first time to investigate the process of infection of human skin by Schistosoma mansoni cercariae. Skin obtained from cosmetic surgery sources was used in the Franz cells to describe the temporal dynamics of the early interaction of cercariae with skin. At 38 degrees C, about 50% of cercariae applied in water to the epidermal surface of the skin were irreversibly attached within 1 min and after 5 min about 85%, were similarly irrecoverable. The technique also provides the means of following the early penetration path of cercariae by histological methods. Franz cell results on the dynamics of attachment/early penetration have been compared with those obtained using artificial skin equivalents and non-human mammalian skin models in the context of the physical and chemical differences between these systems and viable human skin. It is concluded that Franz cells provide a convenient system for directly investigating the early phases of S. mansoni cercariae interaction with human skin.

Ultrastructure of the tegument of the metacercaria of Timoniella imbutiforme.

The spinous body tegument of the metacercaria of Timoniella imbutiforme (Molin, 1859) is described in detail and found to comprise an outer tegumental syncytium connected to subjacently situated subtegumentary 'cells'. There are four types of secretory bodies in the outer syncytial layer as well as serrated overlapping spines and mitochondria. The subtegumentary 'cells' are characterized by the presence of four secretory body types as well as giant bodies which may be involved in the elaboration of the secretory bodies or spine material. The normal configuration of the somatic muscles of T. imbutiforme show that the muscular machinery necessary for activity once the larval stage becomes excysted is already in place. The sensory endings are found to be of the uniciliate type occurring in groups of up to eight in raised domes distributed over the body. Secretory gland cells are numerous and possess long ducts connected to the basal lamina of the outer syncytial layer via septate desmosomes.

Regional specializations of the tegument of the metacercaria of Timoniella imbutiforme.

The ultrastructure of the spinous body tegument of the metacercaria of Timoniella imbutiforme (Molin, 1859) has recently been described. Other regions of the metacercarial tegument, including those of the oral sucker, pharynx, and nephridiopore, demonstrate considerable specializations. The oral sucker tegument had an aspinous outer syncytial layer that possessed a pimpled apical surface as well as enclosing two types of secretory bodies. The pharyngeal tegument likewise lacked spines, but possessed only one type of secretory body, and a smooth but folded outer surface. The nephridiopore tegument, however, showed the greatest degree of specialization possessing a single type of secretory body specific only to this region of the tegument. Also associated with the syncytium here was a prominent long filamentous glycocalyx, and microtubules which were observed for the first time in this region of the tegument.

The parasite community infecting flounders, Platichthys flesus, in the tidal Thames.

The composition of the parasite fauna of the flounder, Platichthys flesus, retrieved from two locations in the tidal Thames is described in detail for the first time. The combined parasite species list of the flounders from Lots Road in the upper tideway and West Thurrock in the middle tideway consisted of one protozoan (Glugea stephani), one monogenean (Gyrodactylus sp.), four larval digeneans (Cryptocotyle concava, Timoniella imbutiforme, T. praeterita, and Labratrema minimus), five adult digeneans (Derogenes varicus, Lecithaster gibbosus, Podocotyle sp., Plagioporus varius, and Zoogonoides viviparus), one larval cestode (unidentified tetraphyllidean), one or possibly more larval nematodes (unidentified) plus five adult nematodes (Capillaria sp., Cucullanus heterochrous, C. minutus, Contracaecum sp. and Goezia sp.), two acanthocephalans (Pomphorhynchus laevis and Acanthocephalus anguillae), three copepods (Lepeophtheirus pectoralis, Acanthochondria sp. and Lernaeocera branchialis), and one mollusc (unidentified glochidia). The overall parasite community of flounders from Lots Road and West Thurrock were compared in terms of species richness and diversity. The parasite community in flounders from the former location in the upper tideway was found to be less diverse than that of its counterpart at West Thurrock in the middle estuary. The component community of Lots Road flounders was dominated by the acanthocephalan Pomphorhynchus laevis.

The use of video-imaging to assess the sub-lethal impact of plant secondary compounds on Schistosoma mansoni miracidia.

The study describes methods developed for using video-imaging technology to record and measure the velocity of Schistosoma mansoni miracidia. The efficacy of the classical bioassay procedure (a qualitative behavioural assay) was compared with that of the new quantitative protocol, for assessing the sub-lethal impact of a larvicidal dichloromethane extract of the seeds of Millettia thonningii on miracidia. The new technique confirmed the efficacy of the classical bioassay for rapid determination of the lethal and sub-lethal impact of larvicides but also provided quantitative information on sub-lethal impacts on miracidial velocity and shape.

Control of water-borne parasitic diseases with natural products: the potential of Dialium guineense as a molluscicide.

The molluscicidal activity of the fruit and leaves of Dialium guineense was found to be due to glycosides of the triterpenoid oleanolic acid. Three glycosides were isolated from the fruit and a fourth from the leaves and are known compounds. The amount of total saponins present in D. guineense makes it a good candidate for a readily available molluscicide in Nigerian villages.

Novel anthelmintic compounds and molluscicides from medicinal plants.

This review assesses the role that can be played by allelochemicals (bioactive secondary compounds) from medicinal and other plants in the control of human helminthic diseases. In the search for new anthelmintics among plant allelochemicals, 3 practical issues have considerable significance. They are the range and capacity of anthelmintic bioassays utilised in preclinical studies in vitro on plant extracts, the phenomenon of coexistent allelochemicals with overlapping activity spectra within single plants, and the problem of non-specific cytotoxins among plant allelochemicals. These topics are discussed in the context of the present absence of any clinically useful plant anthelmintics. In the search for new plant molluscicides for schistosomiasis control, the characteristics of a range of molluscicidal plants are measured against those of the synthetic molluscicide of choice, niclosamide, and against the postulated attributes of practically useful plant molluscicides.

Currently available molluscicides.

Views about the importance of the role of molluscicides in the integrated control of human schistosomiasis have passed through cyclical changes over the past 15 years. For a time, it was hoped that chemotherapy alone would achieve significant morbidity control; it has since become clear that molluscicides cannot be easily excluded from the anti-schistosome armoury. In this review, Sheena Perrett and Phil Whitfield summarize the evidence for this conclusion and provide an overview of currently available synthetic molluscicides and those natural product molluscicides currently under active investigation.

Activity of the molluscicidal plant Millettia thonningii (Leguminosae) toward Biomphalaria glabrata eggs.

A dichloromethane extract of the seeds of the molluscicidal west African legume Millettia thonningii was tested for ovicidal activity toward Biomphalaria glabrata egg masses. The extract was found to be highly ovicidal at concentrations as low as 5 mg/L. Embryonic development of the snails in egg masses was monitored using photomicrographs from which embryonic diameters were estimated. Such measurements revealed that ovicidal effects were developmentally stage specific and normally induced a curtailment of development during the gastrula to trochophore transition.