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Chemo-orientation of echinostome cercariae towards their snail hosts: the stimulating structure of amino acids and other attractants.

The cercariae of Pseudechinoparyphium echinatum and Echinostoma revolutum locate their host snails by turning back when swimming in decreasing gradients of the small molecular weight fraction (< 500) of snail conditioned water. Fractionation and chemical modifications of snail conditioned water from Lymnaea stagnalis showed that amino acids are necessary for the stimulating activity of snail conditioned water. A complete mixture of amino acids in concentrations determined from snail conditioned water had a high attraction. However, differently composed mixtures of amino acids and even single amino acids also had the same attraction as this complete mixture when used in concentrations corresponding to the total concentration of amino acids in snail conditioned water. Experiments with analogues and derivatives of amino acids showed that the primary alpha-amino group and the alpha-carboxyl group are necessary for the full effectiveness of amino acids. The highest effect was elicited by L-amino acids with a primary alpha-amino group, whereas the amino acid type and the chain length seemed to be unimportant. However, the full attraction of snail conditioned water was not achieved by amino acids alone. Chemical modifications of snail conditioned water suggested that the additional stimuli were neither inorganic ions nor organic acids or lipids. As the full attraction of snail conditioned water was obtained when the amino acid mixture of snail conditioned water was combined with its content of urea and ammonia, we conclude that the cercariae use only these excretory products as additional signals for their chemo-orientation. Chemo-orientation to amino acids, urea and ammonia seems to reflect a strategy to locate a broad spectrum of aquatic hosts.

Amino Acids↗

Chemo-orientation of echinostome cercariae towards their snail hosts: amino acids signal a low host-specificity.

The cercariae of Pseudechinoparyphium echinatum and Echinostoma revolutum approach their intermediate host snails by turning back when swimming in decreasing concentration gradients of snail conditioned water. Host stimuli are small molecular amino compounds, probably amino acids. This response was inhibited reversibly when the cercariae were treated with 0.45 microM-silver nitrate. As silver nitrate binds to the ciliated papillae of the cercarial surface, papillar chemoreceptors may be involved. Snail conditioned water from different snail species stimulated different intensities of chemo-orientation in both species of digeneans. However, when the samples of water conditioned with different snail species or even fish, tadpoles and leeches were diluted to the same total amino acid concentrations, the intensities of cercarial responses were similar. Therefore, some specificity of the chemo-orientation seemed to be achieved only by the total concentration of amino acids and not by particular mixtures of amino acids. In fact, amino acid spectra released by freshwater snails varied not only among different snail species, but also within the same species.

Amino Acids↗

Analysis of the gene expression profile of Schistosoma mansoni cercariae using the expressed sequence tag approach.

ESTs constitute rapid and informative tools with which to study gene-expression profiles of the diverse stages of the schistosome life cycle. Following a comprehensive EST study of adult worms, analysis has now targeted the cercaria, the parasite larval form responsible for infection of the vertebrate host. Two Schistosoma mansoni cercarial cDNA libraries were examined and partial sequence obtained from 957 randomly selected clones. On the basis of database searches, 551 (57.6%) ESTs generated had no homologs in the public databases whilst 308 (32.2%) were putatively identified, totaling 859 informative ESTs. The remaining 98 (10.2%) were uninformative ESTs (ribosomal RNA and non-coding mitochondrial sequences). By clustering analysis we have identified 453 different genes. The most common sequences in both libraries represented Sm8 calcium binding protein (8% of ESTs), fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, cytochrome oxidase subunit 1, ATP guanidine kinase and triose phosphate isomerase. One hundred and nineteen identified genes were sorted into 11 functional categories, with genes associated with energy metabolism being the most abundant (13%) and diverse. The diversity and abundance of genes associated with the transcription/translation machinery and with regulatory/signaling functions were also marked. A paramyosin transcript was identified, indicating that this gene is not exclusively expressed in adult worms and sporocysts (as had been suggested previously). The possible physiological relevance to cercariae of the presence of transcripts with homology to calcium binding proteins of the EF-hand superfamily, Gq-coupled rhodopsin photoreceptor, rod phosphodiesterase 8 subunit and peripheral-type benzodiazepine receptor is discussed.

Animals↗

Peroral infection of pigs with Schistosoma japonicum cercariae.

Infections with the zoonotic trematode, Schistosoma japonicum in pigs serves as a valuable model for studying natural definitive host/parasite relationships and a model for human schistosomosis japonica. In the present study the efficiency of a peroral infection route was compared with that of an intramuscular route of infection. Eleven specific pathogen-free Danish Landrace/Yorkshire/Duroc crossbred male and female pigs were divided into two groups of five and six pigs, respectively. Each pig was given 1000 cercariae, either placed in droplets on the mucosa in the buccal cavity, or as medium-suspended cercariae injected into musculus biceps femoris of one of the hindlegs. Ten weeks post infection, all pigs were killed with pentobarbital and the venous system perfused. Worm burdens and liver egg counts were determined and worm fecundity was calculated. S. japonicum infections were established in all individuals in both groups of pigs. When comparing the two groups, the peroral group had significantly higher number of immature worms, whereas the intramuscularly infected group had significantly more worm nodules. However, no difference was seen in total number of worms. No statistical significant differences were found in neither tissue egg counts nor worm fecundity when comparing the two groups. The results from the present study showed a delay in maturation of infection following a peroral infection as compared with an intramuscular infection, but comparability was seen between overall worm establishment and egg production.

Administration, Oral↗

Invasion of skin by Schistosoma cercariae.

Schistosomiasis caused by Schistosoma spp. remains a major global health problem affecting >200 million people. The success of these parasites is a result of their adaptation to several different environments, including snail tissue, fresh water and mammalian blood; and their ability to switch between these environments rapidly. The initial step in infection of the human host involves penetration of the human skin by the aquatic form of Schistosoma, the cercaria. This aspect of host invasion is remarkable because no wounds or insect vectors are required and cercariae can penetrate through intact skin rapidly. The mechanisms of host finding and invasion represent fascinating and complex biological phenomenon, which are discussed here.

Adaptation, Physiological↗

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.

Animals↗

Influence of intramolluscan larval stages of Echinostoma liei on the infectivity of Schistosoma mansoni cercariae.

During co-infection of Biomphalaria glabrata with Schistosoma mansoni and Echinostoma liei, S. mansoni cercariae released before the complete resorption of S. mansoni sporocysts show a very strong decrease of their infectivity in mice. Under conditions of high interspecific competition (i.e. when the snails are infected by E. liei 8 days after infection by S. mansoni), the mean overall worm return is five times lower than that of the control experiment. A marked decrease of the infectivity of cercariae is also noted when snails, infected exclusively with either sporocysts of S. mansoni or rediae of E. liei, are associated in the same tank.

Animals↗

A study of the relationship between temperature and the infectivity of Schistosoma mansoni and Schistosoma haematobium cercariae.

It was shown that maintenance of cercariae of S. mansoni and S. haematobium at low (10degreesC) and high temperatures (40degreesC) markedly increased their mortality during penetration of mammalian host skin. These increased losses in the skin accounted for the known decrease in infectivity, as measured by adult worm recovery, of cercariae kept at low or high temperatures.

Animals↗

The effect of controlled release copper sulphate and tributyltin fluoride on the mortality and infectivity of Schistosoma mansoni cercariae.

Mortality and infectivity or Schistosoma mansoni cercariae were tested against various concentrations of controlled release copper sulphate (EC-8) and tributyltin fluoride (CBL-9B). The results indicate that survival of cercariae varied with concentrations of molluscicide and times of exposures. Statistical analysis reveals that LC50 and LC90 for EC-8 were 1.18 mg/l/hour and 1.82 mg/l/hour while LC50 and LC90 for CBL-9B were 16.8 microgram/l/hour and 21.7 microgram/l/hour respectively. Sublethal concentration of both molluscicides reduced cercarial infectivity considerably. The doses of 0.25 to 0.5 microgram/l/hour for EC-8 and 2.0 to 6.0 microgram/l/hour for CBL-9B were found to be sufficient to inhabit cercarial penetration of the host skin.

Animals↗

Schistosoma mansoni: the occurrence of microvilli on the surface of the tegument during transformation from cercaria to schistosomulum.

Microvilli are developed on the surface of Schistosoma mansoni schistosomula during penetration of the host skin; they form rapidly but are lost approximately 90 min after penetration. Identical microvilli are also formed on schistosomula which have penetrated a mouse skin preparation in vitro, and on schistosomula prepared by mechanical separation of the tail from the body of the cercaria. The microvilli, which are limited by the trilaminate tegumental membrane of the cercaria, eventually degenerate and are cast off from the surface of the tegument. There is little change in the surface area of the schistosomulum at this time, and the formation and loss of microvilli coincides with the replacement of the cercarial tegumental membrane by the new heptalaminate membrane. It is suggested that during the cercaria/schistosomulum transformation, some intramembraneous components of the original cercarial membran e may migrate into the new heptalaminate membrane and thus be retained, while other peripheral components such as the glycocalyx are almost certainly lost.

Animals↗

Diplostomum spathaceum (Rud. 1819): effects of physical factors on the infection of rainbow trout (Salmo gairdneri) by cercariae.

The relationship between infection rates of rainbow trout (Salmo gairdneri) by Diplostomum spathaceum and cercarial concentration, water flow rate and temperature were investigated by means of controlled infections within a flume. A linear relationship was obtained between cercarial concentration and mean abundance of metacercariae/fish. A biphasic relationship occurred between flow rate and abundance of metacercariae. Within the confines of the flume, it was possible to control the infection rate of trout with D. spathaceum cercariae by manipulating flow rate, suggesting that it may be a possible method of controlling diplostomiasis on fish farms. No infection occurred in fish infected and maintained below 10 degrees C and the optimum infection temperature was approximately 17.5 degrees C. Infections became established in fish infected at 7.5 and 5 degrees C but maintained at 15 degrees C prior to examination. Trout were infected at 7.5 degrees C for 10-50 min and all attached cercariae were washed off and removed from the flume. Following infection, fish were either maintained at 7.5 degrees C or 15 degrees C prior to examination. Using this method, it was possible to ascertain that it was migration and not penetration which was inhibited at water temperatures below 10 degrees C.

Animals↗

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.

Calcium Chloride↗

Analysis and comparison of immune reactivity in guinea-pigs immunized with equivalent numbers of normal or radiation-attenuated cercariae of Schistosoma mansoni.

Guinea-pigs immunized with equivalent numbers of normal or radiation-attenuated cercariae of Schistosoma mansoni are known to develop close to complete resistance to reinfection at weeks 12 and 4.5 respectively. We here analyse and compare the immune responses induced by the two populations of cercariae. Both radiation-attenuated and normal parasites of S. mansoni elicited an extensive germinal centre response in guinea-pigs by week 4.5 post-immunization. The anti-parasite antibody titre and cytotoxic activity of serum from 4.5-week-vaccinated, or 4.5-week-infected guinea-pigs were approximately equal, but sera from 12-week-infected individuals had high titres of anti-parasite antibody, which promoted significant larvicidal activity in vitro. In all cases, larvicidal activity was mediated by the IgG2 fraction of the immune serum. Lymphocyte transformation tests conducted on splenic lymphocytes from 4.5-week vaccinated guinea-pigs revealed maximal stimulation against cercarial, 2-week and 3-week worm antigens, whereas spleen cells from 4.5-week-infected guinea-pigs were maximally stimulated by cercarial and 6-week worm antigens. The splenic lymphocyte responses of 12-week infected animals were dramatic against antigens prepared from all life-stages of the parasite.

Animals↗

Behavioural responses of the cercariae of Cryptocotyle lingua (Digenea: Heterophyidae) to computer-controlled shadow sequences.

The cercariae of Cryptocotyle lingua have a brief but active life during which they do not feed. In order to maximize the probability of infection they must respond to a variety of host-related stimuli yet conserve their finite energy reserves by minimizing unnecessary muscular activity. In laboratory experiments, simulated shadows representing the passage of a fish host were found to increase population activity levels. Evidence was also found for a relationship between shadow duration and the duration of subsequent bursts of swimming activity. Adaptation to continuous shadowing was recorded, its onset and magnitude being dependent on both the frequency and duration of shadows delivered. As cercariae aged they became less responsive to shadow stimuli, especially those of short duration.

Animals↗

Evidence that radio-sensitive cells are central to skin-phase protective immunity in CBA/Ca mice vaccinated with radiation-attenuated cercariae of Schistosoma mansoni as well as in naive mice protected with vaccine serum.

Naive CBA/Ca mice and CBA/Ca mice vaccinated 4 weeks previously with radiation-attenuated cercariae of Schistosoma mansoni were subjected to 550 rad of whole body (gamma) irradiation and then challenged 3 days later with normal cercariae. The perfusion recovery data showed that this procedure reduced the primary worm burden in naive mice by 22% and the challenge worm burden in vaccinated mice by 82%. Irradiation also ablated the peripheral blood leucocytes of both mouse groups by 90-100% at the time of challenge. Histological data revealed that such treatment caused a dramatic change in number, size and leucocyte composition of cutaneous inflammatory skin reactions that characterize challenged vaccinated mice and are known to entrap invading larvae; cutaneous eosinophils were preferentially abolished by this treatment. Polyvaccine mouse serum that conferred protection passively upon naive recipient mice, failed to protect naive/irradiated mice when administered by the same protocol. Distraction of macrophages by treatment of mice with silica did not affect the establishment of a primary worm burden and reduced the protection exhibited by vaccinated mice by only 16%. These data indicate that radio-sensitive cells are important to both innate and specific acquired resistance in this mouse model and that macrophages contribute only marginally to the expression of vaccine immunity.

Animals↗

Activated macrophages in highly irradiated cercariae-induced immunity to Schistosoma japonicum in rats.

The results of studies on the schistosomulicidal activity of activated peritoneal and alveolar macrophages (pM phi and aM phi) from rats immunized with highly irradiated (50 krad.) Schistosoma japonicum cercariae are reported. The authors have examined the activation of these macrophages in terms of spreading, adhesion and ingestion of sheep erythrocytes and pinocytosis of horse-radish peroxidase. Using three criteria, peritoneal macrophages and alveolar macrophages from immunized rats and from rats intraperitoneally injected with BCG were significantly more active than those from normal rats or rats stimulated with 10% proteose-peptone or 1% sodium thioglycolate. A significantly higher percentage of adhesion and ingestion was obtained with the sheep erythrocytes that were co-opsonized by heat-inactivated rat anti-sheep erythrocyte serum and fresh normal rat serum. Schistosomulicidal effects were observed with macrophages from irradiated cercariae-immunized rats in two activation systems: in vitro activation in the presence of macrophage-activating factor (MAF), and in vivo activation by the intraperitoneal challenge with sonicated cercarial antigens.

Animals↗

Aerobic to anaerobic transition in the carbohydrate metabolism of Schistosoma mansoni cercariae during transformation in vitro.

Schistosoma mansoni cercariae in water were shown to possess a largely aerobic energy metabolism, the Krebs cycle being the main terminal of carbohydrate breakdown. A metabolic transition towards a more anaerobic breakdown of carbohydrate could be achieved by incubation conditions which also stimulated biological transformation. Incubation of cercariae in a simple salt medium containing 5 mM glucose induced such a metabolic transition: beside carbon dioxide large amounts of lactate and pyruvate were excreted. The results indicate that the production of pyruvate was coupled to electron transfer in the respiratory chain. Some aspects of this unusual pyruvate production are discussed. The observed change in the end-product pattern of carbohydrate breakdown is very rapid: most of the switch occurred within 2 h. Our results show that the metabolic transition was triggered by the biological transformation itself, or by the same event that induces the biological transformation. The metabolic and the biological changes proceeded synchronously.

Animals↗

In vitro release of the anti-gonadotropic hormone, schistosomin, from the central nervous system of Lymnaea stagnalis is induced with a methanolic extract of cercariae of Trichobilharzia ocellata.

Infection with digenetic trematodes causes an inhibition or complete cessation of fecundity in their intermediate hosts, freshwater snails. It has been demonstrated in the host-parasite combination Lymnaea stagnalis-Trichobilharzia ocellata that the action of the female gonadotropic hormones upon their target organs is inhibited by the peptide schistosomin. Schistosomin is produced in the central nervous system of the snail and released upon parasitic infection. In order to study the in vitro release of schistosomin, a bioassay was developed. Central nervous systems were incubated with either an acetic acid or a methanolic extract of larval stages of Trichobilharzia ocellata (miracidia, mother sporocysts, cercariae). The incubation media were chromatographed using HPLC and released schistosomin (-like material) was tested for bioactivity in the calfluxin bioassay. The in vitro release of schistosomin was only induced with a methanolic extract of cercariae. The nature of the cercarial factor is discussed.

Animals↗