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[A stock of infected snails for the mass breeding of cercariae and schistosomules].

A routine to maintain infected snails Biomphalaria glabrata able to produce 1-2 million cercariae per week and a mean shedding of about 3,500 cercariae per snail was quantitatively described. To a monthly production of about 5.3 and 6.5 million cercariae during the years of 1982 and 1983 it was necessary to expose an average of 1,557 and 1,957 snails, respectively. The efficiency described was maintained by infecting 2,000 snails per month (infectivity index of about 60%). A maximal production of 6,000 cercariae/snail was obtained in the period between 56-70 days after the snail infection by miracidia. By this period, however, 90% of the infected snails were dead. In the summer of 1982-1983 it was observed an impairment on the daily cercarial harvests which was related to ambient temperature increase, and the presence of rotifers in the aquaria water. When the cercariae were transformed into schistosomula in vitro, a yield of 55% schistosomula was obtained, with 7% of tail contamination. Lyophilization of cercariae produced 50.9 +/- 63 g dry weight per 1,000 cercariae.

Animals↗

Identification of schistosome-infected snails by detecting schistosomal antigens and DNA sequences.

Cercarial shedding tests do not provide species identification of the schistosomes concerned and cannot detect prepatent schistosomal infections. We have demonstrated that both immunodetection by ELISA of schistosomal antigens in snail hemolymph, and dot hybridization of snail extracts by a DNA probe representing highly repeated sequences, proved suitable for detecting infected snails during prepatency as well as patency. A group-specific monoclonal antibody was found to be suitable for detecting Schistosoma mansoni infection in Biomphalaria sp., but not for positive identification of S. haematobium in Bulinus sp. Comparative evaluation of the diagnostic qualities, and technical aspects and cost of these tests, point to the superiority of the immunodetection approach for large scale detection of snails prepatently infected with S. mansoni. This approach is potentially useful for providing extended information on schistosome-snail epidemiology that may facilitate rapid evaluation of the danger of post-control reinfection, and help make decisions on the time and place of supplementary control measures. In this context the potential usefulness of the immunodetection or DNA probing approach for facilitating catalytic model representation of schistosome-snail epidemiology warrants further evaluation. Specific identification of S. haematobium in Bulinus by either of these approaches may be possible depending on the development of suitable antibodies or DNA probes.

Animals↗

A laboratory-based approach to biological control of snails.

Development of Schistosoma mansoni in the intermediate host Biomphalaria glabrata is influenced by a number of parasite and snail genes. Understanding the genetics involved in this complex host/ parasite relationship may lead to an often discussed approach of introducing resistant B. glabrata into the field as a means of biological control for the parasite. For the snail, juvenile susceptibility to the parasite is controlled by at least four genes, whereas one gene seems to be responsible for adult nonsusceptibility. Obtaining DNA from F2 progeny snails from crosses between parasite-resistant and -susceptible snails, we have searched for molecular markers that show linkage to either the resistant or susceptible phenotype. Both restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) approaches have been used. To date, using a variety of snail and heterologous species probes, no RFLP marker has been found that segregates with either the resistant or susceptible phenotype in F2 progeny snails. More promising results however have been found with the RAPD approach, where a 1.3 kb marker appears in nearly all resistant progeny, and a 1.1 kb marker appears in all susceptible progeny.

Animals↗

Histopathologic features associated with susceptibility and resistance of Biomphalaria snails to infection with Schistosoma mansoni.

Resistance and susceptibility of Biomphalaria snails to Schistosoma mansoni sporocysts occur in different degrees. Histopathology reflects these differences. In a state of tolerance numerous sporocysts in different stages of differentiation are seen in the absence of host tissue reaction. However extensive diffuse and focal proliferation of amebocytes with sequestration and destruction of many parasitic structures appear in resistant snails. Some snails are totally resistant and when exposed to infecting miracidia may never eliminate cercarie. Sequential histopathological examination has revealed that in such cases the infected miracidia are destroyed a few minutes to 24 hr after penetration in the snail. However, B. glabrata that were exposed to S. mansoni miracidia and three months later failed to shed cercariae, exhibited focal and diffuse proliferation of amebocytes in many organs in the absence of pasitic structures. These lesions were similar to those observed in resistant snails that were still eliminating a few cercariae, with the difference that no recognizable sporocystic structures or remnants were present. Histological investigation carried out in similarly resistant B. tenagophila and B. straminea presented essentially normal histologic structures. Only occasionally a few focal proliferative (granulomatous) amebocytic reactions were seen in ovotestis and in the tubular portion of the kidney. Probably, there are two types of reactions to miracidium presented by totally resistant snails: one would implicate the immediate destruction of the miracidium leaving no traces in the tissues; the other involving late reactions that seem to completely destroy invading sporocysts and leave histological changes.

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Molecular identification of symbionts from the pulmonate snail Biomphalaria glabrata in Brazil.

The icthyosporean, Capsaspora owczarzaki, a known predator of Schistosoma mansoni sporocysts in vitro, is more prevalent in laboratory-reared strains of the intermediate snail host, Biomphalaria glabrata resistant to S. mansoni, than from the susceptible M line strain. We examined whether B. glabrata resistant to the NIH-PR-1 strain of S. mansoni from 2 regions in Brazil were also host to C. owczarzaki. Symbiont presence was examined using hemolymph culturing and nested polymerase chain reaction of snail genomic DNA with primers designed to specifically amplify sequences from relatives of the Icthyosporea. All B. glabrata of the resistant Salvador strain from the laboratory of Dr. Lobato Paraense in Rio de Janeiro, Brazil (n = 46) tested negative for symbionts. Three of 18 semiresistant 10-R2 B. glabrata from the laboratory of Dr. Barbosa in Recife, Brazil tested positive for C. owczarzaki. Another icthyosporean, Anurofeca sp., was identified from 1, 10-R2 snail and from 2 of 12 field-collected B. glabrata from Praia do Forte Orange, Ilha de Itamaracá. Snails from 2 other sites, Hotel Colibri, Pontezinha and Praia do Sossego, Ilha de Itamaracá, were negative for Anurofeca. Two genera of ciliates were also identified. Paruroleptus sp. was found in 4, 10-R2 snails and Trichodina sp. was identified in 2 field-collected snails from Praia do Forte Orange and Praia do Sossego.

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Assessment of whole effluent toxicity on aquatic snails: bioaccumulation of Cr, Zn, and Fe, and individual effects in bioassays.

We used a freshwater gastropod, Lymnaea palustris, in chronic bioassays to assess the toxicity of an industrial effluent containing high levels of metals, particularly Cr, Zn, and Fe. Adult snails were exposed for four weeks to different concentrations of effluent sampled at three successive treatment steps (crude effluent, effluent after physicochemical treatment, and after biological treatment). Dose-dependent responses reflecting exposure (metal bioaccumulation) and effects on survival, fecundity, and malondialdehyde production (a proxy for oxidative stress) were investigated. We found that Cr and Zn were accumulated in snail tissues, whereas Fe was regulated. Body concentrations of Cr and Zn decreased along the effluent-treatment gradient, particularly after the physicochemical treatment. For controls versus treatments, no effect on malondialdehyde production was detected. Significant effects were noted for fecundity. The number of eggs per individual decreased for snails exposed to 20, 30, and 40% concentrations of physicochemically treated effluent and for snails exposed to an 80% concentration of the biologically treated effluent. A hormetic effect on the number of eggs per individual was observed for snails exposed to 10 and 20% concentrations of the effluent that had been biologically treated. Deleterious effects of the effluent on L. palustris fecundity were not correlated with high internal concentrations of metals in the snails, suggesting that toxicity resulted from other factors.

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Effect of Echinostoma liei infection on alterations of protein content and some enzymes in Biomphalaria alexandrina snails.

The survival rate and fecundity of B. alexandrina were greatly reduced when exposed to E. liei miracidia. Also, exposure of B. alexandrina snails to E. liei miracidia induced a disruption in the snail metabolism and this effect was more pronounced in 20-days post-miracidial exposure group than in 10-days group. Protein content was significantly reduced in all exposed snail groups than in the control group. A significant elevation in the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymes was recorded also in exposed snails than unexposed ones (control snails). The results indicated also that there were significant increases in the levels of acid and alkaline phosphatases enzymes in exposed snails.

Alanine Transaminase↗

Important aspects of Biomphalaria snail-schistosome interactions as targets for antischistosome drug.

The Biomphalaria species are freshwater snails which have a wide distribution and are significant both medically and economically as intermediate hosts for the schistosome parasite, a digenetic trematode causing schistosomiasis, a disease that infects 200 million people, and domestic animals throughout the tropics. The host-parasite relationship is, in principle, a powerful determinant of the biology of infection and disease. Research on snail-schistosome interactions has the potential for making an important contribution to the study of co-evolution or reciprocal adaptation. The association between Biomphalaria and Schistosoma mansoni could well be an excellent model for studies aimed at elucidating some aspects of the compatibility or resistance of this species to schistosomes. Snail hosts and schistosomes appear to have effects on each other's phenotype and genotype. The objective of this review is to clarify the nature of the relationship between schistosome parasites and their freshwater snail hosts. Aspects of snail-schistosome interactions will be traced in relation to behavioral (growth, reproduction, locomotion), immunological, and biochemical changes induced in the host's tissues by the developing intramolluscan stages of the parasite. This may help to identify biochemical or genetic targets for drug design. Manipulation of the intermediate host through these targets could break the cycle of human and snail infection by schistosomes.

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The host-parasite relationship between the Saudi Arabian Schistosoma mansoni and its intermediate and definitive hosts. 1. S. mansoni and its local snail host Biomphalaria arabica.

The infectivity of miracidia of the Saudi Arabian isolate of S. mansoni in Bi. arabica was found to be influenced by such factors as miracidial dose, water temperature and salinity. The pre-patent period of S. mansoni in Bi. arabica was 30 to 33 days. Miracidial dose had no effect on the mortality of snails during the pre-patent period. The infection rate increased as the miracidial dose was increased. However, cercarial production was highest in snails exposed to 1 miracidium each and decreased as the miracidial dose was increased. Water temperature during exposure had an influence on the mortality, infection rate and cercarial production in Bi. arabica exposed to S. mansoni miracidia. The infection rate was highest in snails exposed at 28 degrees and 34 degrees C. No infection of Bi. arabica occurred at the temperature of 10 degrees C. The number of cercariae per snail per day was highest in snails exposed to miracidia at 34 degrees C. It was demonstrated that salinity had an influence on the infection of Bi. arabica with miracidia of S. mansoni. The infection rate in snails decreased as the salinity increased up to 4,500 mg/l, above which no infection occurred. The daily pattern of cercarial emergence was rhythmic, whereby 94.7% of the total daily production was released within 6 h from infected Bi. arabica, with a peak between 11 a.m. and 1 p.m.

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Schistosoma mansoni: changes in the albumen gland of Biomphalaria glabrata snails selected for nonsusceptibility to the parasite.

The LAC-line strain of the snail Biomphalaria glabrata has very low susceptibility to the parasite Schistosoma mansoni and a very low reproductive potential. Upon examination of the reproductive tract of these snails, light and electron microscopy revealed obvious abnormalities in the albumen gland. Secretory cells that are normally cuboidal in susceptible NMRI (F0) snails were squamous in LAC-line snails. These LAC-line cells contained small secretory granules and negligible rough endoplasmic reticulum and Golgi, compared to large granules and an extensive array of both organelles in F0 albumen gland cells. Comparative analyses of soluble protein extracts of F0 and LAC-line albumen glands showed several qualitative differences. Among the most prominent was an 18-kDa protein in F0 snails that was remarkably reduced in the soluble protein extracts of LAC-line snails. Also, metabolic incorporation of [35S]-methionine was impaired in LAC-line albumen glands. Whether these albumen gland changes are caused by decreased susceptibility to parasitism is yet to determined.

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The development of tissue lesions in the snail Lymnaea glabra exposed to a sublethal dose of molluscicide.

Histological examinations were undertaken in adult Lymnaea glabra to determine whether tissue lesions develop in snails that survived exposure to a molluscicidal agent and thus impair survival or reproduction capacities of remaining snails. The snails were exposed for 4 days to sublethal doses of niclosamide (0.21 mg/L), 3,4-dichloro-2-benzamido-5-nitrothiazole (0.13 mg/L), or 3,5-dichloro-2-benzamido-5-nitrothiazole (0.15 mg/L) at 20 degrees C. After exposure, the surviving snails were maintained under normal conditions (oxygenated water) for a further 21 days. The niclosamide group revealed epithelial necrosis in the digestive glands and the gonads. This was followed by reconstitution from day 12 or 19. The same sequence of tissue lesions also occurred in the kidney, however, a second phase of epithelial necrosis developed in the reconstituted epithelium after day 19. In the two other groups, tissue lesions of the three viscera developed in very similar manner, regardless of the molluscicide chosen for these experiments. Although the molluscicidal doses were sublethal in these snails, 12-19 days at 20 degrees C were required to reconstitute the visceral epithelium. Snails that survived the molluscicidal agent were thus impaired for one week or more in normal conditions.

Animals↗

Effect of X-ray on the snails of schistosomiasis in Egypt.

Biomphalaria alexandrina and Bulinus truncatus snails were exposed to sublethal doses 0.2, 3, 5, 10 and 20 rad of X-ray. The survival and reproductive rates of these snails were highly affected by these doses. The maximum survival periods of laboratory populations of Biomphalaria snails were less than those of field ones which means a high sensitivity of laboratory snails to X-ray. The reproductive capacity of irradiated Biomphalaria and Bulinus snails was highly suppressed and this will interrupt Schistosomiasis transmission. A deleterious effect of gametogenesis of irradiated Biomphalaria was histologically proved. After 3 weeks of snail irradiation with high dose (40 rad) the hermaphrodite gland became completely evacuated.

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PCR amplification of the mitochondrial DNA minisatellite region to detect Schistosoma mansoni infection in Biomphalaria glabrata snails.

Schistosoma infection in Biomphalaria glabrata can be detected by either exposing snails to light to induce cercarial shedding or by squeezing them between glass slides to detect parasites in the digestive gland and other regions. The methods available are inefficient for identification of prepatent infections and do not allow the diagnosis of infection in snails that die before arriving in the laboratory. Furthermore, infection is undetectable after migration of sporocysts from the head-foot region of the snail. We examined the use of polymerase chain reaction (PCR) amplification of minisatellite repeats from Schistosoma mansoni mitochondrial DNA (mtDNA) to identify snail infection. We found that amplification of mtDNA under low stringency conditions (LS-PCR) allowed for the identification of specific S. mansoni infection in Biomphalaria snails. To confirm these results, specific amplification reactions were performed using 2 sets of primers that allowed for the diagnosis of infection and an internal control of the reaction (multiplex PCR). Results obtained using multiplex PCR demonstrated the ability of the assay to detect S. mansoni-specific infection. Thus, LS-PCR as well as specific multiplex PCR allow for the detection of prepatent infections and show high specificity for S. mansoni in comparison with other trematode infections in either living or dead snails.

Animals↗

An evaluation of techniques used in estimating snail populations.

No uniform method, applicable to all situations, can be developed for the quantitative study of bilharziasis vector snail populations. The selection of survey technique and sampling device depends on the objectives of the study, the circumstances in which the work is carried out, the nature of the habitat and the resources available.The various techniques used in obtaining snail-population estimates are divided into two categories-direct methods and indirect methods. The former involve the collection of snails from a specified habitat or for a specified period of time, while the latter include techniques, such as snail marking and palm-leaf traps, in which the snails are not obtained through the efforts of a collector. Each method and device is described in detail, and its suitability under various conditions is discussed.

Animals↗

The behavioural response of slugs and snails to novel molluscicides, irritants and repellents.

The behavioural response of the slug Deroceras panormitanum (Lessona and Pollonera) and the snail Oxyloma pfeifferi (Rossmässler) to novel molluscicides was investigated in choice and no-choice experiments. Low-light video-recording in combination with automated tracking and event recording was used to identify the repellent and irritant effects of (1) cinnamamide, (2) copper ammonium carbonate, (3) a mulch, (4) a horticultural ground-cover matting impregnated with a copper formulation and (5) urea/formaldehyde. In the no-choice experiments the products had a stronger irritant effect on the snails than on the slugs. All products tested except the mulch significantly reduced the locomotor activity of both the slugs and snails. The most effective product, cinnamamide, reduced snail locomotor activity by 94% and track length by 96%. The overall repellent effect of the treatments in the choice experiments was stronger in the slugs; where presence, locomotor activity and track length in the treated area were significantly reduced by all products. The avoidance of treated areas exceeded 95% with the mulch (for slugs) and with copper ammonium carbonate (for snails).

Animals↗

Transfer of cadmium from plant leaves and vegetable flour to the snail Helix aspersa: bioaccumulation and effects.

Juvenile Helix aspersa snails were exposed for 4 weeks to fresh rape leaves (Brassica napus), contaminated by simulating superficial deposits of increasing concentrations of cadmium (Cd). The Cd concentration in leaves was 0.1 in control and 38.4, 93.1, and 177.2 microg x g(-1) (dry mass) in Cd-treated food. The concentration in snail tissues increased with increasing Cd concentrations in the food. The bioaccumulation factors ranged from 4.8 (control) to 2.4 (highest exposed group), indicating a biomagnification of Cd in this food chain. The growth of the snails treated with Cd was reduced by 17, 24, and 43% respectively, compared to the control group. Comparison of these results with those obtained with snails exposed to similar Cd concentrations in a vegetable flour revealed that accumulation and effects were relatively consistent, demonstrating a comparable bioavailability of Cd in the two diets. Tests using growing H. aspersa snails exposed to metals in flour or fresh leaves can be useful for risk assessment purposes.

Animal Feed↗

Protein kinase involvement in land snail aestivation and anoxia: protein kinase A kinetic properties and changes in second messenger compounds during depressed metabolism.

In response to environmental stress (low water, low oxygen) snails sharply suppress their metabolic rate, a process that is coordinated at the molecular level by reversible protein phosphorylation of key enzymes and functional proteins. Factors affecting protein kinase activity are, therefore, critical to metabolic suppression. Changes in the concentration of protein kinase second messenger compounds were followed over the first 24 h of aestivation and anoxia exposure in the terrestrial snail Otala lactea (Muller) (Pulmonata, Helicidae). The results showed declining concentrations of cyclic AMP over the first 24 h of anoxia exposure and aestivation in foot. Cyclic AMP concentrations in hepatopancreas transiently decreased with the lowest concentration observed at 4 h in both anoxic and aestivating animals. A transient increase in foot muscle cyclic GMP concentrations was apparent 4 h after the start of aestivation whereas a slow, steady increase was seen in anoxic foot muscle. Foot muscle 1,4,5-inositol triphosphate (IP3) concentrations decreased transiently during anoxia exposure and aestivation. Hepatopancreas IP3 concentrations were significantly lower in 24 h anoxic snails and foot IP3 concentrations were significantly lower in 24 h aestivating snails. Kinetic characterization of purified PKA catalytic subunit was also performed. Snail PKA catalytic subunit had an absolute requirement for Mg2+ ion but was inhibited at Mg2+ concentrations above 0.5 mM. Increasing concentrations of neutral salts and phosphate also inhibited activity although the inhibition by phosphate appeared to be specific since the inhibition constant (I50 = 39 mM) was much lower than that of the neutral salts (I50 approximately 240 mM). The enzyme exhibited a broad pH optimum between pH 6.5-8.5. Arrhenius plots gave an activation energy of 13.3 kcal/mol corresponding to a Q10 value of 2.3. The relationship between these results and temporal control of enzyme phosphorylation is discussed.

Animals↗

Protein phosphorylation patterns during aestivation in the land snail Otala lactea.

Protein phosphorylation patterns were investigated in whole tissues and subcellular fractions of active and aestivating Otala lactea (Müller) (Pulmonata, Helicidae). Measurement of overall protein phosphorylation showed that incorporation of 32P increased until the second day after injection and remained constant for the remaining 4 days of the time course. Comparison of tissues from aestivating and active snails on day 3 showed a decreased protein phosphorylation in aestivating snails (44% of active). No differences in total and protein-associated radioactivity for foot, mantle or haemolymph were observed. Subcellular fractionation of the hepatopancreas localized the changes to plasma membrane, microsomal, and cytosolic fractions: values for aestivating animals were reduced to 71, 37 and 58% of the corresponding active values. Separation of the individual subcellular fractions on isoelectric focusing columns revealed differences in the phosphate incorporation patterns. Plasma membrane from aestivating animal hepatopancreas had a lower overall level of incorporation and fewer radioactive peaks in the pH 7-10 region than did the plasma membrane fraction from active animals. SDS-PAGE analysis of plasma membrane fractions from active and aestivating snails showed a relative decrease in phosphorylation between 60-80 kDa and 30-40 kDa. IEF analysis of cytosolic proteins from aestivating snail hepatopancreas also showed peaks of radioactivity that were apparently shifted by 0.3 pH units toward higher pI values. Increased phosphate incorporation was observed at a peak that corresponded to the pI value for pyruvate kinase in aestivating snails but definite assignment of peaks was not possible. SDS-PAGE analysis of cytosolic proteins showed an aestivation-related decrease in relative protein phosphorylation between 30-35 kDa and 40-45 kDa. A relative increase in phosphorylation during aestivation was observed for proteins between 16-22 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

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