Evaluation of the Molluscicidal Properties of Euphorbia splendens var. hislopii (N.E.B.) Latex: Experimental Test in an Endemic Area in the State of Minas Gerais, Brazil

Following the positive results obtained regarding the molluscicidal properties of the latex of Euphorbia splendens that were corroborated in laboratory and field tests under restricted conditions, a field study was conducted in experimental streams located in an endemic area. After recording the average annual fluctuations of vectors in three streams, a solution of E. splendens latex at 12 ppm was applied in stream A, a solution of niclosamide at 3 ppm that was applied in stream B and a third stream (C) remained untreated for negative control. Applications of E. splendens and niclosamide resulted in a mortality of 100% among the snails collected in the streams A and B. No dead snails were found in the negative control stream. A monthly follow-up survey conducted during three consecutive months confirmed the return of vectors to both experimental streams treated with latex and niclosamide. This fact has called for a need to repeat application in order to reach the snails that remained buried in the mud substrate or escaped to the water edge, as well as, newly hatched snails that did not respond to the concentration of these molluscicides. Adults snails collected a month following treatment led us to believe that they had migrate from untreated areas of the streams to those previously treated

Identification of Novel Membrane Structures in Plasmodium falciparum Infected Erythrocytes

Little is known about the molecular mechanisms underlying the release of merozoites from malaria infected erythrocytes. In this study membranous structures present in the culture medium at the time of merozoite release have been characterized. Biochemical and ultrastructural evidence indicate that membranous structures consist of the infected erythrocyte membrane, the parasitophorous vacuolar membrane and a residual body containing electron dense material. These are subcellular compartments expected in a structure that arises as a consequence of merozoite release from the infected cell. Ultrastructural studies show that a novel structure extends from the former parasite compartment to the surface membrane. Since these membrane modifications are detected only after merozoites have been released from the infected erythrocyte, it is proposed that they might play a role in the release of merozoites from the host cell

Molecular and Antigenic Characterization of the Leishmania (Viannia) panamensis Kinetoplastid Membrane Protein-11

The kinetoplastid membrane protein 11 (KMP-11) has been recently described in Leishmania (Leishmania) donovani as a major component of the promastigote membrane. Two oligonucleotide primers were synthesized to PCR-amplify the entire coding region of New World Leishmania species. The Leishmania (Viannia) panamensis amplification product was cloned, sequenced and the putative amino acid sequence determined. A remarkably high degree of sequence homology was observed with the corresponding molecule of L. (L) donovani and L. (L) infantum (97% and 96%, respectively). Southern blot analysis showed that the KMP-11 locus is conformed by three copies of the gene. The L. (V) panamensis ORF was subsequently cloned in a high expression vector and the recombinant protein was induced and purified from Escherichia coli cultures. Immunoblot analysis showed that 80%, 77% and 100% sera from cutaneous, mucocutaneous and visceral leishmaniasis patients, respectively, recognized the recombinant KMP-11 protein. In a similar assay, 86% of asymptomatic Leishmania-infected individuals showed IgG antibodies against the rKMP-11. We propose that KMP-11 could be used as a serologic marker for infection and disease caused by Leishmania in America.

Internalization of components of the host cell plasma membrane during infection by Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi attach to the macrophage surface and are internalized with the formation of a membrane bounded vacuole, known as the parasitophorous vacuole (PV). In order to determine if components of the host cell membrane are internalized during formation of the PV we labeled the macrophage surface with fluorescent probes for proteins, lipids and sialic acid residues and then allowed the labeled cells to interact with the parasites. The interaction process was interrupted after 1 hr at 37ºC and the distribution of the probes analyzed by confocal laser scanning microscopy. During attachment of the parasites to the macrophage surface an intense labeling of the attachment regions was observed. Subsequently labeling of the membrane lining the parasitophorous vacuole containing epimastigote and trypomastigote forms was seen. Labeling was not uniform, with regions of intense and light or no labeling. The results obtained show that host cell membrane lipids, proteins and sialoglycoconjugates contribute to the formation of the membrane lining the PV containing epimastigote and trypomastigote T. cruzi forms. Lysosomes of the host cell may participate in the process of PV membrane formation.

The molluscicidal activity of the latex of Euphorbia splendens var. hislopii on Melanoides tuberculata (Thiaridae), a snail associated with habitats of Biomphalaria glabrata (Planorbidae)

The use of the latex of Euphorbia splendens var. hislopii was considered as an effective control method for Biomphalaria glabrata in Sumidouro, Rio de Janeiro. However, the appearance and expansion of the snail Melanoides tuberculata since August 1997, with the concomitant reduction of the population of B. glabrata suggest that competitive exclusion might be taking place. Depending on the susceptibility of the thiarid to the E. splendens toxin, the natural control that is occurring could be interrupted by the employment of the latex if the planorbid were less susceptible to the toxin. The aim of this study is to investigate the molluscicidal activity of the latex on M. tuberculata. We used 420 M. tuberculata, from Sumidouro. Fourteen different latex concentrations were tested using World Health Organization general methodology. Probit analysis was used for LD90 and LD50 determination. The LD50 was 3.57 mg/l and LD90 was 6.22 mg/l. At the highest concentration (10 mg/l) there was no survival. No significant differences among replicas (chi2 = 8.31; gl = 13; p > 0.05) were found. The LD90 dose for M. tuberculata was 13.8 times greater than that for B. glabrata, so that the molluscicide in the presence of the thiarid may have a synergic effect on reduction of Biomphalaria populations.

Molluscicidal action of the latex of Euphorbia splendens var. hislopii N.E.B. ("Christ's Crown") (Euphorbiaceae) against Lymnaea columella (Say, 1817) (Pulmonata: Lymnaeidae), intermediate host of Fasciola hepatica Linnaeus, 1758 (Trematode: Fasciolidae): 1- test in laboratory

The latex action of Euphorbia splendens var. hislopii (Christ's Crown) against snails Lymnaea columella, intermediate host of Fasciola hepatica, derived from irrigation ditches of the Station of Pisciculture at Universidade Federal Rural do Rio de Janeiro, was studied in the laboratory. Lab bioassays, using aqueous solutions of the latex, varying between 0.1 and 10 mg/l, have proven molluscicidal activity of the product collected on the same day the tests were performed, during the four seasons of the year, finding the following lethal concentrations (LC90): 1.51 mg/l in the spring; 0.55 mg/l in the summer; 0.74 mg/l in the fall and 0.93 mg/l in winter, after 24 h exposure of the snails, showing significant differences among the seasons of the year (ANOVA test, F = 11.01, G.L.= 3/33, p < 0.05), as well as among the concentrations (ANOVA test, F = 27.38, G.L.= 11/33, p < 0.05). In the summer, mortality reached 100% from concentration at 0.6 mg/l, the same during fall and in winter as of 1 mg/l, while in spring it only reached 100% mortality as of 2 mg/l. Mortality in the controls was low, reaching 5% in the summer and winter and 10% in the fall and spring. None of the samples died. During the assay, with an aqueous solution of the latex at a concentration of 5 mg/l, in order to check the time of duration of the product effect, in the laboratory, it was observed that the molluscicidal activity remained stable up to the 15th day after the beginning of the test with 100% mortality of L. columella, gradually losing its effect until the 23rd day, when we no longer observed animal mortality. In the control group, there was a random daily variation in mortality rate ranging 0-50% after 48 h of observation for 30 days.

Activity of Euphorbia splendens var. hislopii N.E.B. (Euphorbiaceae) latex against Lymnaea columella (Say, 1817) (Pulmonata: Lymnaeidae), intermediate host of Fasciola hepatica, Linnaeus, 1758 (Trematoda: Fasciolidae). 2: limited field-testing

The molluscicidal evaluation of Euphorbia splendens var. hislopii (Crown of thorns) against Lymnaea columella snails, intermediate host of Fasciola hepatica, in irrigation ditches of the Pisciculture Station at Universidade Federal Rural do Rio de Janeiro, was studied under limited field conditions. An aqueous solution of the latex at 5 mg/l was tested in two irrigation ditches (experimental and control ditches), after initial sampling of the snail population present. Twenty-four hours after application of the product, it was verified that 97.4% of free L. columella snails and 100% of snails of the same species captive in cages and used as sentinels at three points equidistant from the application site in the experimental ditch, died. For Biomphalaria tenagophila and Melanoides tuberculata snails, present in the experimental ditch, the mortality was 100%, for the species Pomacea spp. the mortality was 40%. No mortality was verified in the free mollusks, or in the sentinels in the ditch used as control. E. splendens var. hislopii latex is thus an efficient natural molluscicide, which may be used as an alternative control agent against L. columella.

Studies of membrane fluidity and heart contractile force in Trypanosoma cruzi infected mice

In Chagas disease serious cardiac dysfunction can appear. We specifically studied the cardiac function by evaluating: ventricle contractile force and norepinephrine response, affinity and density of beta-adrenergic receptors, dynamic properties of myocardial membranes, and electrocardiography. Albino swiss mice (n = 250) were infected with 55 trypomastigotes, Tulahuen strain and studied at 35, 75, and 180 days post-infection, that correspond to the acute, indeterminate, and chronic phase respectively. Cardiac beta-adrenergic receptors' affinity, myocardial contractility, and norepinephrine response progressively decreased from the acute to the chronic phase of the disease (p < 0.01). The density (expressed as fmol/mg.prot) of the receptors was similar to non-infected mice (71.96 ± 0.36) in both the acute (78.24 ± 1.67) and indeterminate phases (77.28 ± 0.91), but lower in the chronic disease (53.32 ± 0.71). Electrocardiographic abnormalities began in the acute phase and were found in 65% of the infected-mice during the indeterminate and chronic phases. Membrane contents of triglycerides, cholesterol, and anisotropy were similar in all groups. A quadratic correlation between the affinity to beta-adrenergic receptors and cardiac contractile force was obtained. In conclusion the changes in cardiac beta-adrenergic receptors suggests a correlation between the modified beta-adrenergic receptors affinity and the cardiac contractile force.

Physiological changes in Biomphalaria glabrata say, 1818 (Pulmonata: Planorbidae) caused by sub-lethal concentrations of the latex of Euphorbia splendens var. hislopii N.E.B (Euphorbiaceae)

Molluscides have been used as one of the strategies to control schistosomiasis. Many plant extracts with molluscidal effects have been tested, but the action of the latex of Euphorbia splendens var. hislopii is considered the most promising because it meets the recommendations of the World Health Organization (WHO). The objective of this study was to determine the lethal dose and identify the effects of the different doses of latex of E. splendens var. hislopii on the physiology of Biomphalaria glabrata submitted to treatment for 24 h. The concentrations of glucose, uric acid and total proteins in the hemolymph and of glycogen in the digestive gland and cephalopodal mass were determined. The LD50 value was 1 mg/l. The highest escape index was found to be at a concentration of 0.6 mg/l. The results showed that the latex of E. splendens var. hislopii caused a sharp reduction in the reserves of glycogen in the digestive gland and elevation of the protein content in the hemolymph of B. glabrata.

Latex constituents from Calotropis procera (R. Br.) display toxicity upon egg hatching and larvae of Aedes aegypti (Linn.)

Calotropis procera R. Br. (Asclepiadaceae) is a well-known medicinal plant with leaves, roots, and bark being exploited by popular medicine to fight many human and animal diseases. This work deals with the fractionation of the crude latex produced by the green parts of the plant and aims to evaluate its toxic effects upon egg hatching and larval development of Aedes aegypti. The whole latex was shown to cause 100% mortality of 3rd instars within 5 min. It was fractionated into water-soluble dialyzable (DF) and non-dialyzable (NDF) rubber-free materials. Both fractions were partially effective to prevent egg hatching and most of individuals growing under experimental conditions died before reaching 2nd instars or stayed in 1st instars. Besides, the fractions were very toxic to 3rd instars causing 100% mortality within 24 h. When both fractions were submitted to heat-treatment the toxic effects were diminished considerably suggesting low thermostability of the toxic compounds. Polyacrylamide gel electrophoresis of both fractions and their newly fractionated peaks obtained through ion exchange chromatography or desalting attested the presence of proteins in both materials. When submitted to protease digestion prior to larvicidal assays NDF lost most of its toxicity but DF was still strongly active. It may be possible that the highly toxic effects of the whole latex from C. procera upon egg hatching and larvae development should be at least in part due to its protein content found in NDF. However the toxicity seems also to involve non protein molecules present in DF.

Reproductive activity alterations on the Biomphalaria glabrata exposed to Euphorbia splendens var. hislopii latex

The reproductive activity of Biomphalaria glabrata exposed to Euphorbia splendens var. hislopii latex was evaluated. Parameters related to fecundity and fertility were observed. The snails were exposed to the LD50 (1 mg/l) of crude latex. At the first week post exposure (p.e.), the egg laying was reduced. After the fourth week p.e., an increase of the number of eggs/snail occurred. The results showed a marked reduction in the hatching of the snails, revealing an interference of latex exposure with the reproductive process of B. glabrata of E. splendens var. hislopii. The LD50 of the latex may be used as an alternative method to control the size of the populations of B. glabrata in field.

Transferrin uptake may occur through detergent-resistant membrane domains at the cytopharynx of Trypanosoma cruzi epimastigote forms

Uptake of transferrin by epimastigote forms of the protozoan Trypanosoma cruzi occurs mainly through a cytostome/ cytopharynx, via uncoated endocytic vesicles that bud off from the bottom of the cytopharynx. We have here examined whether detergent-resistant membrane (DRM) domains might be involved in this process. Purified whole cell membrane fractions were assayed for cholesterol levels and used in dot blot analyses. Detergent-resistant membrane markers (cholera B toxin and anti-flotillin-1 antibody) presented positive reaction by dot blots in cholesterol-rich/ protein-poor membrane sub-fractions. The positive dot blot fraction was submitted to lipid composition analysis, showing composition similar to that of raft fractions described for other eukaryotic cells. Immunofluorescence assays allowed the localization of punctual positive signal for flotillin-1, matching the precise cytostome/ cytopharynx location. These data were confirmed by immunofluorescence assays with the co-localization of flotillin-1 and the transferrin uptake site. Our data suggest that DRM domains occur and are integrated at the cytostome/ cytopharynx of T. cruzi epimastigotes, being the main route for transferrin uptake.

Kinetoplastid membrane protein-11 is present in promastigotes and amastigotes of Leishmania amazonensis and its surface expression increases during metacyclogenesis

Kinetoplastid membrane protein-11 (KMP-11), a protein present in all kinetoplastid protozoa, is considered a potential candidate for a leishmaniasis vaccine. A suitable leishmaniasis vaccine candidate molecule must be expressed in amastigotes, the infective stage for mammals. However, the expression of KMP-11 in Leishmania amastigotes has been a subject of controversy. We evaluated the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, of Leishmania amazonensis by immunoblotting, flow cytometry and immunocytochemistry, using a monoclonal antibody against KMP-11. We found that KMP-11 is present in promastigotes and amastigotes. In both stages, the protein was found in association with membrane structures (at the cell surface, flagellar pocket and intracellular vesicles). More importantly, its surface expression is higher in amastigotes than in promastigotes and increases during metacyclogenesis. The increased expression of KMP-11 in metacyclic promastigotes, and especially in amastigotes, indicates a role for this molecule in the parasite relationship with the mammalian host. The presence of this molecule in amastigotes is consistent with the previously demonstrated immunoprotective capacity of vaccine prototypes based on the KMP-11-coding gene and the presence of humoral and cellular immune responses to KMP-11 in Leishmania-infected humans and animals.

Carbohydrate metabolism alterations in Biomphalaria glabrata infected with Schistosoma mansoni and exposed to Euphorbia splendens var. hislopii latex

This paper evaluates the alterations in the glycogen content of tissues (digestive gland and cephalopedal mass) and glucose in the haemolymph of Biomphalaria glabrata BH strain infected with Schistosoma mansoni BH strain and exposed to the latex of Euphorbia splendens var. hislopii. A reduction in the glycogen deposits was observed in infected snails exposed and not exposed to latex. However, the exposure to latex caused a greater depletion of the glycogen levels in both sites analysed, especially from the third week onward. The utilisation of latex as a molluscicide to control the population of infected B. glabrata selectively is proposed.