Emergence of cercariae of Echinostoma caproni and Schistosoma mansoni from Biomphalaria glabrata under different laboratory conditions

Release of Echinostoma caproni cercariae and Schistosoma mansoni from experimentally infected Biomphalaria glabrata snails maintained under different laboratory conditions was studied. Infected snails were isolated individually for 1 h in Stender dishes containing 5 ml of artificial spring water and the number of cercariae released during this time was recorded. Of numerous conditions tested, the addition of lettuce, the use of water conditioned by B. glabrata snails and a temperature of 35 degrees C significantly increased the release of E. caproni cercariae. A significant increase in cercarial release of S. mansoni was seen only in cultures fed lettuce. A temperature of 12 degrees C caused a significant decrease in cercarial release of both E. caproni and S. mansoni. Increased snail activity associated with feeding behaviour was probably responsible for the enhanced cercarial sheds observed in this study.     

Shedding patterns of Schistosoma mansoni and Ribeiroia marini cercariae from a mixed infection of Biomphalaria glabrata

When the snail Biomphalaria glabrata is doubly infected by two species of trematode, Schistosoma mansoni and Ribeiroia marini, each species keeps its own cercarial shedding pattern. Emergence of S. mansoni cercariae occurs during the photophase while the R. marini cercariae are shed during the night. These results demonstrate the absence of interference between the mechanisms responsible for the cercarial shedding of each species. The only difference noted concerns a two-hour shift in the peak emergence of S. mansoni as a consequence of the antagonism between the two parasites.     

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.     

Effect of the organophosphorous insecticide, chlorpyrifos (Dursban), on growth, fecundity and mortality of Biomphalaria alexandrina and on the production of Schistosoma mansoni cercariae in the snail.

Exposure of Biomphalaria alexandrina to sublethal concentrations (0.125, 0.25 and 0.05 ppm) of the organophosphorous insecticide, chlorpyrifos (Dursban), induced a reduction in egg production and egg hatchability. Exposure of Schistosoma mansoni miracidia to the insecticide (60 min, 0.50 ppm) prior to infection of B. alexandrina did not affect the subsequent production of cercariae. However, exposure of S. mansoni-infected snails to the insecticide until day 55, from day 20 to day 62 and from day 35 to 62 following infection resulted in blockage of cercarial shedding. Cercarial shedding commenced in some snails when the treatment stopped. Exposure to the insecticide in concentrations of 0.125 and 0.25 ppm during the first 20 days following infection did not affect the subsequent production of cercariae, but exposure to 0.5 ppm during the first 20 days affected markedly the production of cercariae due to a high snail mortality. The findings indicate that the cercaria is the target stage for the activity of chlorpyrifos on the intramolluscan larval development. It is suggested that S. mansoni cercarial production in B. alexandrina may be a useful system for monitoring the effect of low concentrations of pesticides on the aquatic environment, and that the ability by chemical means to interrupt the cercarial production might be a useful tool in further analyses of important aspects of the snail/parasite relationship.     

Schistosoma mansoni: inhibition of cercarial "penetration" proteases by components of mammalian blood.

1. Normal human sera and plasma were fractionated in order to identify inhibitors of the "penetration" proteases of Schistosoma mansoni cercariae. 2. The main inhibitor, accounting for 90% of the total activity of serum, appears to be alpha 1-antitrypsin (alpha 1-AT) as identified by separation on DEAE-cellulose and Sephadex, by immunoelectrophoresis and by anticercarial protease activity of purified alpha 1-AT preparations. 3. The inhibition profiles of purified preparations of the 6 known serum antiproteases suggest that the parasite protease is similar to vertebrate chymotrypsin. 4. On a molar basis, the order of inhibitory activity against the cercarial protease is: alpha 1-AT = alpha 2-macroglobulin; C'-1-inactivator; alpha 1-antichymotrypsin. No inhibition was obtained with inter-alpha-inhibitor or antithrombin III.     

Oxygen uptake and lactate production by Schistosoma mansoni cercaria, cercarial body and tail, and schistosomule.

1. Oxygen consumption by Schistosoma mansoni cercarial bodies varies, with the batch of organisms, the incubation media and the temperature (27-37 degrees C), from 27.4 +/- 3.4 to 55.0 +/- 4.8 microliters O2/mg larval protein per hr. It is proportional to the concentration of organisms incubated, up to 25,000/ml, as calculated from whole protein. 2. Oxygen uptake by cercariae is inhibited by 5.6 mM glucose in the incubation media, a concentration that stimulates the respiration of cercarial bodies. 3. No significant differences in the oxygen uptake were presented by cercarial bodies with and without glycocalyx or glandular secretions, or devoid of all of them. 4. Inhibitors of the Krebs cycle and the respiratory chain, and uncoupling agents influence the oxygen uptake by cercariae, cercarial bodies and schistosomules to the same extent. 5. The permeability change presented by transformed larvae had no influence on the excretion of lactate by cercarial bodies, which is about 0.3 mumoles/mg protein per hr and remains constant for 5 hr; under nitrogen, this amount increased 70%. Cercariae in anaerobiosis, however, excreted as much as 15 times more lactate than under air. 6. Lactic dehydrogenases of cercariae, cercarial bodies and tails, and schistosomules are of the muscle type and do not change during the transformation.     

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.     

Effects of alterations in the heartbeat rate and locomotor activity of Schistosoma mansoni-infected Biomphalaria glabrata on cercarial emergence

During a 27-hr period of food deprivation, Biomphalaria glabrata infected with Schistosoma mansoni had lower heartbeat rates and locomotor activities than did controls that were feeding ad lib. However, there was no difference between the cercarial emergence from control and experimental snails either before, during, or after the period of food deprivation. No correlation was found between the locomotor activity of the snail and the number of cercariae emerging from the snail in continuous light. Our results indicated that the emergence of S. mansoni cercariae from B. glabrata was not affected by the heartbeat rate or locomotor activity of the snail. The factors controlling the rhythm of S. mansoni cercarial emergence from B. glabrata may be independent of the snail.     

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.     

Cercarial chaetotaxy and sex differentiation of Schistosoma mansoni deriving from humans and Nectomys squamipes (Muridae: Sigmondontinae) in Brazil

A comparative study was made between sympatric isolates of Schistosoma mansoni: one from a wild rodent (R) Nectomys squamipes and another one from humans (H) isolated from a low endemic schistosomiasis transmission area in Brazil. Our purpose was to detect differences between them concerning chaetotaxy (number and pattern of distribution of the argentophilic papillae) of the cercariae by means of silver impregnation. No significant difference (x > 0.05) between isolates was noted. Nevertheless, a significant difference (x < 0.05) was observed in the cercarial index (ratio of the distance between the first and the second preacetabular papillane and the distance between the first and the second dorsal preacetabular papillae) of male and female cercariae in both isolates. Males presented a greater cercarial index than females. By means of multivariate analysis, male cercariae were distinguished from female cercariae through the following characteristics: average number of dorsal papillae on the right quadrant, average number of ventral middle papillae on the right quadrant (H isolate) and average number of dorsal middle papillae on the left quadrant (R isolate). The results suggest that R and H isolates belong to the same population that could complete its life cycle in rodent-snail-rodent fashion.     

Effect of some pharmacological substances on the motility of the Cryptocotyle lingua cercaria (Heterophyidae)

The effect of some biologically active substances (acetylcholine, serotonin, octopamine, sodium nitroprussid and FMRF-amide) on the motility of the Cryptocotyle lingua cercariae was studied. Solutions of FMRF-amide, octopamine, and sodium nitroprussid have no statistically significant influence on the motility of C. lingua. Acetylcholine and serotonin in solutions affected the motility through the prolongation of the active phase of swimming. Further research is required to elucidate the mechanisms underlying the cercarial motility.     

Resistance to desiccation of Biomphalaria glabrata adults infested by various miracidia of Schistosoma mansoni

1200 adult Biomphalaria glabrata were submitted during 6 weeks to anhydrobiosis condition. Some snails were healthy, some were previously infected 3 days or 12 days ago with 8 +/- 2 miracidia of Schistosoma mansoni, others were shedding cercariae. The snails were put on soil or buried into hermetically closed, or ventilated, plastic boxes. There was no survival of snails kept in sealed boxes, or among positive snails, but 44% of control healthy snails and 40.6% of infected (for 3 or 12 days) snails in ventilated boxes were living at the term of the desiccation stage. Survival was better for "on soil" snails than for "buried" snails, but no difference was shown between 3-days and 12-days infection. The surviving desiccated B. glabrata had a lesser death rate and a lesser cercarial production than infected snails kept in water. An inferior production of male cercariae comparatively to female and to "mixed" cercariae was demonstrated by statistical analysis of the cercarial sheddings. In all positive snails, periodic variations of cercarial production was shown, whatever the sex of those cercariae. In addition many pauses of the sheddings were established by the authors.     

Detection of Schistosoma mansoni circulating cathodic antigen for evaluation of resistance induced by irradiated cercariae.

The appearance of serum levels of circulating cathodic antigen (CCA) detectable by a monoclonal antibody (mAb) (5H11) antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) system was evaluated during acute Schistosoma mansoni infections in female CF1 mice exposed to either 100 or 25 cercariae. Measurable CCA levels occurred in these groups at 5 and 7 wk after infection, respectively. The kinetics of appearance of CCA were thus related to the intensity of infection. The level of resistance developed by female C57BL/6 mice upon immunization with irradiated cercariae, as expressed by both worm burden and CCA levels after cercarial challenge was evaluated. Immunization conferred 44% protection against the challenge infection, and the level of CCA detected in the sera of the control group was significantly (P less than 0.02) higher than that found in the sera of the immunized group, 6 wk after challenge. These results demonstrate that CCA detection by the 5H11 mAb antigen-capture sandwich ELISA can reflect vaccine-induced resistance against S. mansoni. Localization studies showed that 5H11 reacts with a CCA epitope in the adult worm gut and to a lesser extent with the male tegument. Adaptations of this and other antigen detection systems may prove useful in monitoring the efficacy of developmental vaccines, an ability that may be essential for the extension of such studies to humans.     

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.     

Factors affecting surface changes in intact cercariae and cercarial bodies of Schistosoma mansoni.

The effect of different incubation media and of temperature on the induction of water sensitivity has been investigated in intact and tailless Schistosoma mansoni cercariae. Removal of the cercarial tail by vortex stirring and elevation of the temperature of the medium from 27 to 37 degrees C resulted in the rapid onset of permeability changes in the larvae. The rate of change was greater in water than in TC-199 or Hanks' BSS media. Lowering the pH of the medium or increasing the concentration of Ca2+ ions decreased the rate of permeability change: raising the pH of the medium or the addition of 10(-5) M EDTA enhanced the rate. Raising the temperature of the medium also increased the rate of permeability change in intact cercariae although the rates obtained varied with the different media tested, being greatest in TC-199. It is concluded that both temperature elevation and loss of the cercarial tail influence the onset and rate of permeability changes in cercarial bodies during the transformation to schistosomula.     

The H3 receptor is involved in cholecystokinin inhibition of food intake in rats.

We investigated the peripheral effects of an H3-receptor agonist and an H3-receptor antagonist (R)alpha-methylhistamine (Ralpha-MeHA) and thioperamide, respectively, on basal feeding and the CCK8-induced inhibition of food intake in rat. Intraperitoneal injection of thioperamide reduced food intake in a dose-dependent manner with maximal inhibition (35%, P<0.01 vs saline) at 3 mg/kg. (R)alpha-MeHA (0.3-3 mg/kg i.p.), an H3-receptor agonist alone had no effect on feeding but reversed the thioperamide-induced inhibition of food intake in a dose-dependent manner. The maximal feeding inhibitory dose of thioperamide (3 mg.kg i.p) increased by 40% and 22 % (P<0.01 vs saline) brain and stomach histamine contents, respectively. Histamine (0.3 - 6 mg/kg i.p.) and CCK-8 (3 - 30 microg/kg i.p) also inhibited food intake in a dose-dependent manner. Inhibition was 20% to 40% for histamine and 40% to 80% (P<0.01 vs saline) for CCK8. CCK-8 inhibition of feeding was increased by thioperamide and prevented by (R)alpha-MeHA in a dose-dependent way. In addition, CCK-8 did not reduce food intake if rats were pretreated with pyrilamine or ranitidine postsynaptic H1- and H2-receptor antagonists respectively. Our data suggest that the H3-receptor is involved in basal feeding. They also suggest that CCK satiety depends upon the release of histamine which acts on the H2- and H1-receptors, the final mediators of this effect.     

Arresting tissue invasion of a parasite by protease inhibitors chosen with the aid of computer modeling

Computer modeling of the three-dimensional structure of an enzyme, based upon its primary sequence alone, is a potentially powerful tool to elucidate the function of enzymes as well as design specific inhibitors. The cercarial (larval) protease from the blood fluke Schistosoma mansoni is a serine protease hypothesized to assist the schistosome parasite in invading the human circulatory system via the skin. A three-dimensional model of the protease was built, taking advantage of the similarity of the sequence of the cercarial enzyme to the trypsin-like class of serine proteases. A large hydrophobic S-1 binding pocket, suspected from previous kinetic studies, was located in the model and confirmed by new kinetic studies with both synthetic peptide substrates and inhibitors. Unexpected structural characteristics of the enzyme were also predicted by the model, including a large S-4 binding pocket, again confirmed by assays with synthetic peptides. The model was then used to design a peptide inhibitor with 4-fold increased solubility, and a series of synthetic inhibitors were tested against live cercariae invading human skin to confirm that predictions of the model were also applicable in a biologic assay.     

Schistosoma mansoni: influence of Biomphalaria glabrata size on susceptibility to infection and resultant cercarial production

Biomphalaria glabrata snails of the same age, but different sizes, were used to determine size-related susceptibility to Schistosoma mansoni miracidial infection and the influence of snail size on total cercarial production. Snails with shell diameters from less than 5 to greater than 17 mm were individually exposed to one or several miracidia, depending on the experiment. In snails exposed to multiple numbers of miracidia, the percentage of snails which developed patent infections was lower in snails with larger shell sizes. This was also reflected by fewer primary sporocysts per infected snail found in tissues of the larger snails. Upon determining cercarial production in these groups over a 1-month period there were no statistical differences between any groups in the numbers of cercariae produced per snail. However, upon determining the number of successful primary sporocysts found in cohort snails of each size group, cercarial production increased as a function of the number of successful primary sporocysts. This was verified by examining cercarial production in various size snails with known monomiracidial infections. Our data therefore confirm and extend earlier work using snails infected with unknown numbers of miracidia and clearly show that total S. mansoni cercarial development and decreased susceptibility of snails is a direct reflection of snail size and not necessarily age of the snail.     

Sequential histological changes in Biomphalaria glabrata during the course of Schistosoma mansoni infection

Biomphalaria glabrata, highly susceptible to Schistosoma mansoni, were seen to shed less and less cercariae along the time of infection. Histological examination kept a close correlation with this changing pattern of cercarial shedding, turning an initial picture of no-reaction (tolerance) gradually into one of hemocyte proliferation with formation of focal encapsulating lesions around disintegrating sporocysts and cercariae, a change that became disseminated toward the 142nd day post miracidial exposure. Findings were suggestive of a gradual installation of acquired immunity in snails infected with S. mansoni.     

Schistosoma mansoni: in vitro conversion of cercariae to schistosomula.

Schistosoma mansoni cercariae were incubated for 3-5h at 37 degrees C in various test solutions, and the rate and extent of conversion of the cercariae to schistosomula were determined. Criteria used to identify schistosomula included: (1) loss of cercarial tail, (2) viability of organisms in saline but not in water, (3) pre-acetabular gland evacuation and (4) ability to survive in culture. Incubation of cercariae in rat chamber fluid resulted in organisms which were water sensitive, but retained their tails and pre-acetabular gland contents. Conversion to water sensitivity was not blocked by 0-01 M EDTA.