Schistosoma mansoni: correlations between mouse strain, skin eicosanoid production, and cercarial skin penetration

We have previously reported that cercarial penetration is highly correlated with cercarial production of leukotrienes (LT's) and hydroxyeicosatetraenoic acids (HETE's). Because skin also produces various eicosanoids, we undertook an investigation of skin eicosanoids in various strains of mice and 1 strain of rat in order to ascertain if skin eicosanoids could be correlated to cercarial penetration. SENCAR, ICR, NMRI, A/J, C3H/HeJ, C57Bl/6, ASEBIC, and BALB/c mouse strains were used in this study as well as the SD-Rat strain. The ability of cercariae to penetrate skin was strain specific. A/J and SENCAR mice had the highest penetration rates (approximately 98%), whereas the SD-Rat strain had the lowest (43%). These penetration rates we linearly correlated with tail skin HETE production at 10 min (R = 0.826), whereas HETE production at 60 min had a parabola-shaped relationship (R = 0.793). The primary infection of mice with Schistosoma mansoni cercariae may therefore be directly correlated with both the skin's innate ability to synthesize HETE, as well as with cercarial eicosanoid production, especially HETE levels. However, we believe that skin eicosanoid production is just one of many factors affecting cercarial skin penetration. Other factors discussed are: skin surface fatty acid levels, cercarial eicosanoid production, epidermal vs. dermal eicosanoid production, and the immunocompetence of the host.     

Host identification by Schistosoma japonicum cercariae

Attachment, the first phase of host identification by Schistosoma japonicum cercariae, can occur in 2 different ways. Cercariae clinging to the water surface simply swing around and transfer to the host skin. Free-swimming cercariae behave like S. mansoni: upon touching a substrate, they switch from tailward to forward movement, swim in an arc, and attach to it with the penetration organ. Neither type of attachment is influenced by chemical, thermal, or specific mechanical stimuli from the host. The second phase, remaining on the host, requires a solid hydrophobic surface and seems to depend only on the cercaria's ability to cling to it. This phase is not influenced by chemical or thermal stimuli. The third phase, creeping across the host surface, is independent of chemical and mechanical stimuli. Cercariae migrate in thermal gradients to a preferred temperature of 37 +/- 3 C and then attempt to penetrate. Penetration, the fourth phase, was evoked by human skin surface lipids. The free fatty acid (FA) fraction was identified as exclusively stimulating components. Saturated FA's were effective at chain lengths between 10 and 14 carbon atoms (pH 7.0), and unsaturated FA's were effective at longer chains and their activity increased with increasing number of double bonds. Dog skin surface components did not stimulate cercarial penetration, which can be attributed to the lack of free FA's. A temperature of 32-40 C also stimulated penetration responses, which might be the main stimulus in animal hosts, whose skin surfaces contain no or only a few free FA's. FA's and heat evoked a transformation of cercarial tegument simultaneous with penetration behavior, making the organisms osmotically sensitive. The host identification of S. japonicum cercariae is very nonspecific compared with the differentiated host recognition of S. mansoni.     

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.     

Schistosoma mansoni: pH dependence of cercarial eicosanoid production, penetration, and transformation

The pH dependence of Schistosoma mansoni cercarial penetration and transformation was determined using a gelatin:agar matrix, containing 3mM linoleate, as a penetration substrate. Penetration was largely unaffected by pH, approaching 100% over the pH range of 5.4 to 8.2, while transformation had an optimum pH range between 6.2 and 7.4. Within this pH range, between 74 and 89% of cercariae lost their tails. Outside this range, transformation decreased to 0% above pH 7.8 and dropped to 57% at pH 5.4. Esculetin, a lipoxygenase inhibitor, also incorporated into the agar:gelatin plates at a concentration of 1 mM, had little effect on cercarial penetration, except between pH 6.5 and 6.65, where penetration rates fell to 50% at pH 6.63. Transformation, however, was inhibited by esculetin, except between pH 6.5 and 6.8, where transformation was statistically equivalent to controls (P = 0.064, two-tailed Student's t test). Cercarial eicosanoid production measured at pH 6.55 and 7.2 in the presence and absence of 1 mM esculetin has led to the tentative identification of a 5-lipoxygenase product associated with cercarial penetration: LTB4 or 6-trans LTB4, a breakdown product of LTB4. We discuss the importance of pH control in cercarial experiments as well as the possible modulatory role skin pH (surface, epidermal, and dermal) may have in regulating cercarial eicosanoid production.     

A protein with lectin activity in penetration glands of Diplostomum pseudospathaceum cercariae

Homogenates of Diplostomum pseudospathaceum cercariae agglutinated mouse erythrocytes. The haemagglutination could be inhibited by certain glycoconjugates containing beta-1,3- and beta-1,4-glycan chains and also by some simple saccharides. The most potent inhibitors were heparin and some other glycosaminoglycans, bacterial lipopolysaccharides, laminarin (a beta-1,3-glucan) and lactulose. After electrophoresis of cercarial proteins, a dominant double band appeared in the 22-24 kDa region of gels. On blots, this protein bound labelled laminarin and it was also one of the few proteins recognised by mouse antibodies raised against cercarial haemagglutinins. In addition, mouse polyclonal antibodies against the beta-1,3-glucan-binding protein bound exclusively to the 22-24 kDa region on Western blots. Histochemistry revealed strong binding of labelled laminarin to cercarial penetration glands; this reaction was fully blocked by unlabelled laminarin. Other labelled glycoconjugates such as heparin, hyaluronic acid and a bacterial lipopolysaccharide also bound to the glands. Immunohistochemistry confirmed the localisation of the beta-1,3-glucan-binding protein in penetration glands. Reaction of the cercarial protein with immunoglobulins from non-immunised mice was observed on both nitrocellulose membranes and histological sections; this could be blocked by laminarin in incubation buffers. We consider the cercarial haemagglutinin to be a lectin which is identical with the 22-24 kDa beta-1,3-glucan-binding protein. According to the binding specificity and localisation we speculate on a role of this lectin in cercarial penetration into the host, probably as a tissue recognition or antibody rendering factor.     

Partial purification and characterization of an inhibitor from newborn-rat epidermis with activity against the proteinase of Schistosoma mansoni cercariae.

The penetration of cercariae through the skin initiates infection of the host with the human trematode parasite Schistosoma mansoni. Many larvae fail to migrate into the living epidermal cell layer. In order to determine if chemical as well as mechanical barriers to cercarial skin penetration exist, inhibitory activity of epidermal cell extracts against the proteinase obtained from cercarial secretions was assayed. An inhibitor was purified 50-fold by gel filtration on Sephadex G 75 and cation exchange chromatography at pH 5.8 and 4.9. The inhibitor has a relative molecular mass (Mr) of approx. 40 000-53 000. Oxidation of the inhibitor with N-chlorosuccinimide eliminated its inhibitory activity and thus indicated a critical methionine residue. The inhibitor was active against a wide spectrum of serine proteinases: porcine pancreatic elastase, human granulocyte elastase, bovine trypsin, and bovine alpha-chymotrypsin. However, no inhibition was detected against papain or clostridial collagenase. The inhibitor did not cross react with antiserum to human or rat serum alpha 1-proteinase inhibitor.     

The role of essential fatty acids and prostaglandins in cercarial penetration (Schistosoma mansoni)

We used rat skin membranes to test the putative role of prostaglandins (PG) and essential fatty acids (EFA) in the penetration response of Schistosoma mansoni cercariae. To examine the effects of EFA on cercarial penetration an EFA-deficient rat model was used. Dams were fed an EFA-deficient diet during lactation and the pups were weaned to this diet. Cercarial penetration of EFA-deficient rat skin membranes was not reduced from control levels until 12 wk on the diet. At this time a decrease of 64.3% was observed. This decrease remained constant up to 16 wk, after which the study was terminated. Other normal rats were treated with 20 mg/kg ibuprofen, a PG inhibitor, to examine the role of PG in the penetration response. Treated rat skin contained a mean of 2 micrograms ibuprofen per 30 mm3 of skin (25-mm skin disc) at 1.5 hours post-injection. Skin from treated rats inhibited penetration by over 81%. These studies indicate that skin EFA and PG may have a critical role in the completeness of penetration by cercariae through the skin, although it is not clear whether cercarial or host PG are involved in the penetration response.     

Schistosoma mansoni: production of cercarial eicosanoids as correlates of penetration and transformation

A method was developed, using a 0.25% agar matrix, to incorporate varying concentrations of linoleate and correlate cercarial transformation and eicosanoid production in vitro. Schistosoma mansoni cercariae were stimulated to penetrate over a wide range of linoleate concentrations; however, the transformation process occurred over a narrow range. Approximately 25% of cercariae penetrated the agar matrix in controls (no linoleate) and 0.003 mM linoleate. Penetration rates rose gradually until, at linoleate concentrations of 0.3 mM or greater, penetration approached 100%. The transformation process did not begin until the linoleate concentration in agar reached 2.0 mM (3.8%), and achieved maximum (91%) at 3.0 mM. A concentration of 9.0 mM linoleate gave 100% penetration and transformation rates, but penetration was superficial and cercariae were not viable. Cercarial eicosanoid production was concentration-related. Various eicosanoid classes were associated with cercarial penetration and transformation. Penetration rates were correlated with increasing leukotriene (LT, R = 0.9541) and hydroxyeicosatetraenoic acid (HETE, R = 0.8363) levels, while transformation rates correlated with increasing prostaglandin levels (R = 0.9225). Correlating eicosanoid production with penetration and transformation rates strengthened the hypothesis that successful cercarial penetration and transformation are dependent on both skin essential fatty acid levels and resulting cercarial eicosanoid production.     

Delayed tail loss during the invasion of mouse skin by cercariae of Schistosoma japonicum

A traditional assumption is that schistosome cercariae lose their tails at the onset of penetration. It has, however, recently been demonstrated that, for Schistosoma mansoni, cercarial tails were not invariably being shed as penetration took place and a high proportion of tails entered human skin under experimental conditions. This phenomenon was termed delayed tail loss (DTL). In this paper, we report that DTL also happens with S. japonicum cercariae during penetration of mouse skin. It occurred at all cercarial densities tested, from as few as 10 cercariae/2·25 cm(2) of mouse skin up to 200 cercariae. Furthermore, it was demonstrated that there was a density-dependent increase in DTL as cercarial densities increased. No such density-dependent enhancement was shown for percentage attachment over the same cercarial density range.     

Complement-mediated tail degradation of Neodiplostomum seoulense cercariae

The furcocercus cercariae of Neodiplostomum seoulense (Digenea: Neodiplostomidae) penetrate the skins of tadpoles and shed their tails. The speculated mechanism of this tail loss was physical efforts required to produce a vigorous zigzag motion during skin penetration; no other mechanism has been proposed. We examined the relationship between the host serum and cercarial tail loss. Cercariae of N. seoulense were collected from experimentally infected Segmentina hemisphaerula, and lots of 300 cercariae were cultured in medium 199 contained several types of sera. Cercarial tail degradation was induced in all media, but all the cercariae cultured except those cultured in media containing fetal bovine serum (FBS) died within 48 hr. After 72 hr cultivation in media containing FBS, cercarial tail degradation was induced in 67.0%; in continuous cultivation 13.3% of larvae survived for 7 days. Tail degradation did not occur in the absence of serum and when serum was heat inactivated at 56 degrees Celsius for 30 min. The addition of 20 mM ethylenediaminetetraacetic acid (EDTA) blocked cercarial tail degradation completely. Moreover, the addition of 20 mM MgCl2 restored tail degradation blocked by EDTA. These results suggest that the alternative complement pathway is related with the N. seoulense cercarial tail degradation induced by serum.     

The role of prostaglandins in cercarial (Schistosoma mansoni) response to free fatty acids

In examining the structure-activity relationship of a diverse group of chemicals reported to prevent cercarial penetration after topical application, we noticed a moiety that was common to free fatty acids and prostaglandins. Because unsaturated fatty acids have been reported to stimulate cercarial penetration, we hypothesized that cercarial stimulation by skin and fatty acids may invoke prostaglandin mechanisms in cercariae, skin, or both. Thus we compared the stimulation of cercariae by a series of essential and nonessential fatty acids and demonstrated an inhibition of this response by ibuprofen and aspirin, known cyclo-oxygenase inhibitors, and by 13-azaprostanoic acid, a potent antagonist of the thromboxane/endoperoxide receptor. These data led us to postulate a major role for prostaglandins in the cercarial penetration response.     

Physiological analysis of cercarial behavior.

The behavior of trematode cercariae was made accessible to physiological analyses by splitting the continuous flow of behavior into separate units of behavior patterns. Such phases include active, passive, and resting phases in the intermittent swimming mode, attachment to the host, remaining on the host, directed creeping to suitable entry sites, and penetration phases including penetration movements, tail shedding, tegument transformation, and secretion of enzymes. Each of these phases may be stimulated by separate environmental and host signals. The pattern of the responses and the chemical, thermal, mechanical, and visual stimuli have been described in some detail but only a few studies have dealt with the question of how these responses are coordinated by receptors and nervous systems. Highly specific and sensitive chemoreceptors for host signals such as carbon dioxide, L-arginine, fatty acids, and glycoproteins have been defined from cercarial behavior, but they have not yet been allocated to morphological structures. Analyses of cercarial behavior of 6 schistosomatid and 4 fish-infecting species revealed that the individual parasite species show different behavioral patterns and respond to very different host signals, even though they infect congeneric hosts. The adaptive benefits of such behavioral diversity and complexity still are to be elucidated.     

Schistosoma mansoni: experimental chemoprophylaxis in mice using oral anti-penetration agents

The ability to prevent schistosomiasis by using an oral chemoprophylactic agent, which acts by preventing cercarial penetration, has been unexplored. We initially examined the effect of praziquantel (PZQ) as such an agent and found that it was moderately effective in blocking cercarial penetration, but that this effect was dependent on the vehicle used to administer the drug. ICR mice were given a total of 200 mg/kg PZQ per os over an 8-hr period in a divided dose of 50 mg/kg/2 hr. At time periods ranging from 0 to 92 hr after the last dose, mice were exposed to approximately 75 75Se-labeled cercariae via the tail. Twenty-four hours later, mice were sacrificed, their tails were removed and subjected to autoradiography, and the percentage of penetration was calculated. Cremophor El, 50% PEG 200, 50% propylene glycol, vegetable oil, and cod liver oil were used as PZQ vehicles. When Cremophor El (ethoxylated castor oil) was used to administer PZQ, a 93% reduction in cercarial penetration was seen at 0 hr and a 98%+ reduction rate was seen from 4 to 24 hr postexposure. However, Cremophor El alone had an essentially equivalent effect on cercarial penetration from 8 to 92 hr after administration. These unexpected results led us to investigate both castor oil and ricinoleic acid (castor oil is 87% ricinoleate as triglyceride) as oral anti-penetration agents. Mice were given the lipids orally by gavage for 7 days. On Day 8, each group of 12 mice was exposed to approximately 75 75Se-radiolabeled cercariae. Castor oil gave protection rate ranging from 90 to almost 100% at an optimal concentration of 0.3 ml/day x 3 or 7 days (approximately 9.8 g/kg/day). These observations suggest that chemoprophylaxis may be possible by dietary supplementation with lipids having anti-penetration activity or by molecules that resemble these lipids.     

Changes in survival characteristics of Diplostomum spathaceum cercariae emerged from cadmium-exposed Lymnaea stagnalis

The effect of exposing Lymnaea stagnalis (Gastropoda: Pulmonata), infected with Diplostomum spathaceum (Trematoda: Diplostomatidae), to 100 microg l(-1) cadmium for 7 days on survival characteristics (survival, tail loss, decaudized cercarial life-span) of emerged cercariae was investigated. Exposure of L. stagnalis to cadmium resulted in significantly increased D. spathaceum cercarial survival and an inhibited tail loss compared to controls. The normal parallel relationship which exists over time between decreasing cercarial survival and increasing tail loss in controls was changed in cercariae from cadmium-exposed hosts with an increased proportion of cercarial deaths occurring without tail loss. The decaudized cercarial life-span over the survival period of the cercarial population did not significantly change. However comparisons between individuals decaudized during the initial 24 h time period with those which were decaudized during the final period of cercarial survival showed a significantly altered life span which did not occur in the control population. As a potential indicator of penetration 'fitness' comparisons were also undertaken between control and exposed cercariae decaudized during the initial 24 h time period, which revealed that the decaudized cercarial life-span from the exposed hosts was significantly different from controls. This may have important implications for the ability of cercariae to migrate through the tissues of their target host. The importance and relevance of these results to parasite transmission are discussed.     

Purification and properties of a proteolytic enzyme from the cercariae of the human trematode parasite Schistosoma mansoni.

Skin penetration by the cercarial stage of the human trematode parasite Schistosoma mansoni is mediated by the secretion of proteolytic enzymes able to digest components of mammalian connective tissues. In the present study the purification of these proteinases from cercarial homogenates is reported. The major proteinase species has a mol. wt. of approx. 25 000 and exists in monomeric form as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This proteinase has an isoelectric point of 6.0. Studies presented here, with a variety of substrates and inhibitors, confirm previous claims that these proteinases belong to the serine class, and, in addition, suggest that they resemble the vertebrate chymotrypsins rather than trypsins or elastases. However, the amino acid composition of the cercarial proteinase differs significantly from bovine chymotrypsin and from the human leucocyte chymotrypsin-like cathepsin G. The amino-acid-composition differences between these proteinases are consistent with their differences in isoelectric point. In order to obtain an insight into the role of the proteinase in skin penetration, its activity on cartilage proteoglycan monomers and on the isolated peptide backbone of proteoglycan was studied. The results of the present study indicate that the cercarial enzyme catalyses a limited specific digestion of the peptide core.     

On the development of body-wall musculature Diplostomum chromatophorum metacercariae (Trematoda: Diplostomidae)

The body-wall musculature of invasive cercariae and metacercariae of Diplostomum chromatophorum at different intervals after the penetration into the experimental intermediate host Cyprinus carpio (1, 3, 5, 6, 7, 10, 12, 20, 34, 40 days) has been investigated with the help of TEM technique. During the first 10 days after the invasion (in conditions of our experiment), the cercarial subtegumental muscle fibres degenerate. These muscles are replaced by newly formed ones. Mass differentiation of myoblasts beneath the tegument was observed in 7-10-day-old metacercariae. Obtained data indicate the metamorphosis of body-wall musculature during the morphogenesis in Diplostomum chromatophorum metacercariae.     

Schistosoma mansoni: eicosanoid production by cercariae

Cercariae of Schistosoma mansoni are stimulated to penetrate skin by certain free fatty acids. The cercariae have an active arachidonate cascade, presumably using host skin essential fatty acids as cascade precursors. Exposing cercariae to 3.3 mM linoleate for 1, 10, and 60 min resulted in production of a wide variety of eicosanoids. Using high-performance liquid chromatography, eicosanoids coeluting with prostaglandin E2, D2, and A2, leukotriene B4, and 5-hydroxyeicosatetraenoic acid standards were identified, as well as unidentified peak positions. Radioimmunoassay confirmed the presence of immunoreactive prostaglandin E1, and E2, and 5- and 15-hydroxyeicosatetraenoic acids in cercarial extracts. No eicosanoid production occurred when cercariae were exposed to 3.3 mM oleate and 1 or 330 microM linoleate. Both high-performance liquid chromatography and radioimmunoassay data indicated that cercariae regulate the production of eicosanoids through time. It is postulated that arachidonate metabolism and subsequent eicosanoid production are required for successful cercarial penetration.     

The physiology of itch

The perception of itch is associated with many parasites and their vectors, especially following penetration of the skin by the parasites themselves, as in cercarial dermatitis of schistosome infections, or penetration of arthropod mouthparts during blood feeding. Many ectoparasites such as scabies, lice and fleas, provoke sensations of itch - even when the insects are no longer (or have never been) present, giving rise to the phenomenon of delusory parasitosis. Itch, and the host 'grooming' responses with which it is associated, is increasingly recognized as an important factor in modulating vector feeding behaviour, which can have profound effects on the transmission dynamics of vector borne parasites. As a background to future reviews of this developing subject, we asked John Alexander, author of the classic Arthropods and Human Skin (Springer-Verlag, 1984), to explain what is itch, and to discuss what is known about its underlying Physiology.     

Virulence factors of schistosomes

This review considers whether the products of schistosomes in the mammalian host can be considered as virulence factors. These include: the cercarial secretions used in infection, those of the migrating schistosomulum, surface-exposed proteins of adult worms in the portal system and their gut vomitus in the context of immune evasion, secretions of the egg facilitating its escape from gut tissues and micro-exon gene products.