Destruction of Schistosoma mansoni sporocysts in Biomphalaria glabrata after phytochemical exposure.

Schistosomiasis is a neglected tropical disease and affects over 200 million people worldwide. The snail Biomphalaria glabrata is one of the intermediate hosts of S. mansoni. The aim of this work was to verify the action of Euphorbia milii var. hislopii latex in the hemocytes profile and histopathology of B. glabrata infected by S. mansoni. Uninfected and infected snails were exposed to sublethal concentration of E. milii latex for 24 hours (1.0 mg/L). The survival rate was 88.5% for the uninfected snails and 66.6% for the infected and exposed snails. In the snails infected by S. mansoni, the exposure to E. milii latex promoted proliferation of hemocytes in the tentacles, mantle, digestive gland and kidney. In the digestive gland and the kidney, granulomatous reactions occurred around the sporocysts and caused their destruction. The number of circulating hemocytes from the group infected and exposed to E. milii latex was significantly higher than in the other groups. Three types of hemocytes were found: hyalinocytes, granulocytes and blast-like cells. We conclude that the E. milii latex influenced the cellular immune response of the susceptible B. glabrata strain to infection by S. mansoni, promoting the destruction of parasites.

Behavioral response of Biomphalaria glabrataexposed to a sublethal concentration of Euphorbia miliivar. hislopii latex.

The Euphobia milii var. hislopii latex has been tested in the control of schistosomiasis but its action in the locomotor activity of Biomphalaria glabrata is unknown. The objective of this work was to study the locomotor and reproductive behaviors of B. glabrata exposed to E. milii var. hislopii latex. For this, 96 snails were individually exposed to the latex (LC50 - 0.53 mg / L) for 24 hours. The specimens were submitted to biomonitoring for image analysis to record the locomotor parameters at different times: before exposure (control), one day post exposure (group 1 d-p-e) and 30 days post exposure (group 30 d-p-e). The reproductive parameters were recorded weekly for 10 weeks. All locomotor activities of group 1 d-p-e decreased significantly. The egg/egg mass ratio decreased the week after exposure while there was an increase in the hatching rate. After 30 days, these reproductive parameters were similar to those observed in these same snails before exposure. However, the number of hatched snails declined after exposure until the end of the experiment. The influence of the latex in the parameters of B. glabrata added further evidence that this natural water-soluble product can be an important tool for the control of schistosomiasis.

Changes in the locomotory and reproductive behavior of Biomphalaria glabrata infected with Schistosoma mansoni.

The infection and development of a parasite may cause physiological, morphological and behavioral changes in its host. Changes in the locomotory activity of a host induced by their parasites may also influence the life-cycles of both host and parasite in the environment. The aim of the present work was to evaluate the locomotory activities of Biomphalaria glabrata before and after an experimental infection with Schistosoma mansoni relating to the shedding of cercaria. In addition, the reproductive parameters of infected B. glabrata were analyzed during the prepatent and patent periods of the infection. The locomotory activity was recorded using an image analysis biomonitoring system based on a Videomex V. Five parameters were analyzed: 'Distance traveled', 'Ambulatory time', 'Stereotypic time', 'Resting time' and 'Average speed'. The number of shed cercariae was counted twice at 45 and 52 days post-infection. The reproductive parameters of infected B. glabrata analyzed were the numbers of egg masses, eggs and hatched snails. All statistical analyses were performed using the R program. Of the 69 snails infected with S. mansoni, 33 (47.8%) shed cercariae ('positive') and 36 (52.2%) ('exposed') failed to exhibit any cercarial shedding prior to the end of the experiment. The locomotory activity of the all snails increased significantly after infection with S. mansoni. However, when the 'positive' and 'exposed' snails were compared, the former, shedding cercariae, were less motile. With regard to reproduction, 84.8% (28/33) of the 'positive' and 27.7% (10/36) of the 'exposed' snails failed to lay egg masses during patent period. The number of cercariae individually shed by each 'positive' snail presented a positive relation with 'Stereotypic time' and a negative relation with egg laying. Our findingshighlight the way in which infection with S. mansoni affects the locomotory and the reproductive behavior of B. glabrata. The number of cercariae shed is directly associated with the reduction/interruption in egg-laying and with an increase in random movement.

Biological, biochemical and histopathological features related to parasitic castration of Biomphalaria glabrata infected by Schistosoma mansoni.

Parasitic castration in the snail-trematode relationship can be understood as any change in the reproductive function of the snail that is due to interference by the developing larvae inside the snail that leads to the reduction or complete disruption of egg-laying activity. This study was designed to observe the parasitic castration of Biomphalaria glabrata infected with Schistosoma mansoni during both the pre-patent and patent periods. The effect of infection on snail fecundity and fertility, growth rate and survival was studied during the 62 days following miracidia exposure. An integrated approach was employed that used biochemical and histological tools over the same period. To study the effect of infection on reproduction, we individually exposed 30 snails to 5 miracidia each and tracked their fertility and fecundity. For our histopathological studies, 50 snails were exposed to 20 miracidia each, and for our histochemical studies, 50 snails were exposed to 5 miracidia each. An equal number of uninfected snails were used as a control for each group. The B. glabrata exposed to the BH strain of S. mansoni showed 50% positivity for cercarial shedding. Both the experimental and control groups showed 100% survival. The pre-patent period lasted until 39 days after exposure to miracidia. Exposed snails that showed cercarial shedding exhibited higher growth rates than either exposed snails that did not demonstrate cercarial shedding or uninfected controls. Exposed snails without cercarial shedding and uninfected controls showed no differences in the reproductive parameters evaluated during the patent period; snails experiencing cercarial shedding showed a reduction in fecundity and fertility. These snails began to lay eggs only after the 50th day post miracidia exposure. The haemolymph glucose levels showed an oscillating pattern that decreased during periods of greater mobilisation of energy by the larvae and was accompanied by a depletion of glycogen in the cephalopodal mass and digestive gland. Histopathological examination at 55 days showed that the ovotestis was highly atrophied. There was almost complete disappearance of germ cells, and the supporting stroma formed a nearly empty net. At day 45, the infected digestive gland showed a high cylindrical epithelium with little preserved cytoplasm. The contents of the secretory granules of the albumen gland of infected animals stained with Alcian blue (AB), pH 1.0, indicating the presence of sulphated carbohydrates. Thus, parasitic castration in the B. glabrata-S. mansoni model may be regulated directly and indirectly by the developmental stage of the trematode and the biochemical and histopathological alterations during the patent period of infection.

A rapid diagnostic test for Schistosomiasis mansoni.

This article presents an improvement to the Kato-Katz (KK) method, making it faster and more efficient for the visualisation of fertile eggs in stool samples. This modified KK method uses sodium acetate formalin as a fixative and reveals the intensity of infection in less than 1 h, reducing the diagnostic time without increasing the cost. This modified method may contribute to future epidemiological studies in both hospitals and the field due to its rapid and precise diagnostic, which allow for immediate treatment.

The Effect of Different Formulations of Praziquantel in Reducing Worms in the Prepatent Period of Schistosomiasis in Murine Models.

Schistosomiasis is a widely distributed parasitic disease and one of the most important neglected tropical diseases globally, for which Praziquantel® (PZQ) is the only available treatment. In this context, tests with new PZQ formulations become relevant for disease control. This study evaluated the effects of PZQ treatment in the prepatent phase of schistosomiasis using two formulations: nanoencapsulated (PZQ-NANO) and active pharmaceutical ingredient (PZQ-API). Five experimental groups were established, for which the following serological parameters were evaluated: ALT, AST, ALP, and TP. Animals treated with PZQ-API at 15 and 30 days post-infection showed decreased eggs per gram of feces (EPG) compared to untreated infected animals. The same animals showed reductions of 63.6 and 65.1%, respectively, at 60 days post-infection. Animals treated with PZQ-NANO experienced no significant changes in EPG at any time of observation. Animals treated with either PZQ-API or PZQ-NANO had higher ALT and AST levels in the patent period (60 and 90 days post-infection). Treatment with PZQ, either API or NANO, at 15 days post-infection reduced AST, ALT, and TP levels. It is concluded that prepatent treatment with PZQ-API can reduce the parasite load of infected animals and that treatment at 15 days post-infection can prevent increased serum levels of ALT, AST, and TP.

Elemental Contamination in Brown Mussels (Perna perna) Marketed in Southeastern Brazil.

Perna perna mussels, abundant throughout the Brazilian coast, are routinely applied as bioindicators in environmental monitoring actions due to their sessile and filter-feeding characteristics. In addition, they are noteworthy for their food importance, especially for coastal populations. In this context, the aim of this study was to investigate elemental contamination in commercially marketed and highly consumed P. perna samples from the highly impacted Guanabara Bay, Rio de Janeiro, Brazil. A total of 30 mussels were sampled, and elemental concentrations (As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, V, and Zn) were determined in adductor muscle samples by inductively coupled plasma mass spectrometry (ICP-MS). Human consumption risks were assessed by comparisons to Brazilian and international legislations. No significant differences between sex were observed for all analyzed elements. Even when analyzing only the adductor muscle, all mussel samples exceeded the Brazilian limit for Cr, while 12 samples exceeded the limit for Se. When compared to other regulatory agencies, As and Zn levels were higher than the limits set by China, New Zealand, and the USA. Estimated daily dietary intake values were not above limits imposed by the Food and Agriculture Organization of the United Nations/World Health Organization for any of the assessed elements, although it is important to note that only the adductor muscle was assessed. Therefore, continuous metal and metalloid monitoring in bivalves in the study region is suggested, as metal transport and bioavailability, especially in coastal estuaries such as Guanabara Bay, which are currently undergoing significant changes due to anthropogenic activities.

Influence of Echinostoma paraensei (Lie and Basch, 1967) infection on the calcium content in Biomphalaria glabrata (Say, 1818).

The calcium content in the hemolymph and shell of Biomphalaria glabrata (Say, 1818) was determined after exposure to different parasite burdens (5 and 50 miracidia) of Echinostoma paraensei (Lie and Basch, 1967). The snails were dissected 1, 2, 3, and 4 weeks after infection to collect the hemolymph and shell. An increase in calcemia was observed in snails infected with both miracidial doses. A significant decrease in the calcium ions in the shell was observed, coinciding with the calcemia peak in the hemolymph. This indicates greater mobilization of calcium between the shell and hemolymph to regulate the calcium content in the body when the snail is exposed to stress conditions, as has also been observed in some other infected snail species. The results obtained indicate that in this model, the calcium metabolism depends on the miracidial dose used.

Changes in the reproductive biology of Biomphalara glabrata infected with different doses of Echinostoma paraensei miracidia.

The egg-laying rate, number of egg masses, number of eggs/mass, number of eggs hatched/snail and egg viability of Biomphalaria glabrata exposed to different doses (5 and 50) of Echinostoma paraensei miracidia were analyzed as indicators of reproductive activity. Polystyrene plates were placed in aquariums containing the snails and every other day for four weeks after infection the plates were removed to count the number of egg masses and eggs laid. After this, the plates were numbered individually and placed in new aquariums free of snails and the egg masses were observed daily to determine the hatching rate. On average there was an increase in the parameters evaluated in the infected snails in relation to the controls (uninfected snails), except for egg viability, which was significantly lower in the groups infected with 50 miracidia. These findings indicate that when infected, this host snail is able to increase its reproductive activity, suggesting an ecological strategy to maintain the species.

Feasibility of near-infrared spectroscopy for species identification and parasitological diagnosis of freshwater snails of the genus Biomphalaria (Planorbidae).

Near Infrared Spectroscopy (NIRS) has been applied in epidemiological surveillance studies of insect vectors of parasitic diseases, such as the Dengue's mosquitoes. However, regarding mollusks, vectors of important worldwide helminth diseases such as schistosomiasis, fascioliasis and angiostrongyliasis, NIRS studies are rare. This work proposes to establish and standardize the procedure of data collection and analysis using NIRS applied to medical malacology, i.e., to mollusk vectors identifications. Biomphalaria shells and live snails were analyzed regarding several operational aspects, such as: moisture, shell side and position of the live animal for acquisition of NIR spectra. Representative spectra of Biomphalaria shells and live snails were collected using an average of 50 scans per sample and resolution of 16 cm-1. For shells, the sample should first be dried for a minimum of 15 days at an average temperature of 26±1°C, and then placed directly in the equipment measurement window with its left side facing the light beam. Live animals should be dried with absorbent paper; placed into a glass jar, and analyzed similarly to the shells. Once standardized, the technique was applied aiming at two objectives: identification of Biomphalaria using only the shells and parasitological diagnosis for Schistosoma mansoni infection. The discrimination of the three Biomphalaria species intermediate hosts of S. mansoni only by shell has technical limit due to the scarcity of organic material. Nevertheless, it was possible to differentiate B. straminea from B. tenagophila and B. glabrata with 96% accuracy. As for the parasitological diagnosis, it was possible to differentiate infected mollusks shedding S. mansoni cercariae from the non-infected ones with 82, 5% accuracy. In conclusion, the Near Infrared Spectroscopy (NIR's) technique has proven to be an innovative and sound tool to detect infection by S. mansoni in the different species of Biomphalaria intermediate hosts.

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.

Phytochemical Molluscicides and Schistosomiasis: What We Know and What We Still Need to Learn.

Worldwide schistosomiasis remains a serious public health problem with approximately 67 million people infected and 200 million at risk of infection from inhabiting or transiting endemically active regions. Africa, South America, the Caribbean, and the Middle East are the main transmission regions of Schistosoma mansoni. The fight against transmission through the use of molluscicides is not recent and has been advocated as the only activity with the possibility of interruption of transmission in small, epidemiologically active outbreaks. Euphorbia milii var. hislopii (syn. splendens) (Des Moulins, 1826) is the most promising for use in official schistosomiasis control programs according to the WHO. In this review, we show that an understanding of some how E. milii latex affects the snail vector and their parasites from a molecular level to field conditions is lacking. On the other hand, this type of treatment could also provide a rationale for the control of schistosomiasis and other parasitosis. Several publications contribute to enforcing the use of E. milii latex in endemic countries as a cheap alternative or complement to mass drug treatment with praziquantel, the only available drug to cure the patients (without preventing re-infection).

Double impact: natural molluscicide for schistosomiasis vector control also impedes development of Schistosoma mansoni cercariae into adult parasites.

BACKGROUND: Schistosomiasis has been reported in 78 endemic countries and affects 240 million people worldwide. The digenetic parasite Schistosoma mansoni needs fresh water to compete its life cycle. There, it is susceptible to soluble compounds that can affect directly and/or indirectly the parasite's biology. The cercariae stage is one of the key points in which the parasite is vulnerable to different soluble compounds that can significantly alter the parasite's life cycle. Molluscicides are recommended by the World Health Organization for the control of schistosomiasis transmission and Euphorbia milii latex is effective against snails intermediate hosts. METHODOLOGY/PRINCIPAL FINDINGS: We used parasitological tools and electron microscopy to verify the effects of cercariae exposure to natural molluscicide (Euphorbia milii latex) on morphology, physiology and fitness of adult parasite worms. In order to generate insights into key metabolic pathways that lead to the observed phenotypes we used comparative transcriptomics and proteomics. CONCLUSIONS/SIGNIFICANCE: We describe here that the effect of latex on the adult is not due to direct toxicity but it triggers an early change in developmental trajectory and perturbs cell memory, mobility, energy metabolism and other key pathways. We conclude that latex has not only an effect on the vector but applies also long lasting schistosomastatic action. We believe that these results are of interest not only to parasitologists since it shows that natural compounds, presumably without side effects, can have an impact that occurred unexpectedly on developmental processes. Such collateral damage is in this case positive, since it impacts the true target of the treatment campaign. This type of treatment could also provide a rational for the control of other pests. Our results will contribute to enforce the use of E. milii latex in Brazil and other endemic countries as cheap alternative or complement to mass drug treatment with Praziquantel, the only available drug to cure the patients (without preventing re-infection).

The influence of exposure to Euphorbia splendens var. hislopii latex on the concentrations of total proteins and nitrogen products in Biomphalaria glabrata infected with Schistosoma mansoni.

The aim of this work was to analyze the content of total protein and nitrogen degradation products in Biomphalaria glabrata infected with Schistosoma mansoni and exposed to Euphorbia splendens var. hislopii latex. The LC(50) of this latex was 1.0mg/l. Concentrations of uric acid, urea and total proteins were determined in the hemolymph of B. glabrata infected with five S. mansoni miracidia and exposed to a sublethal concentration of E. splendens var. hislopii latex for 24h. The exposure to this molluscicide caused total depletion of the alterative sources of energy (total proteins) and significant variation in the nitrogen degradation products. The urea content increased while the uric acid level decreased. These results reflect a disturbance in the snails regulation of their metabolism due to intoxication caused by the latex exposure.

A rapid diagnostic test for schistosomiasis mansoni

This article presents an improvement to the Kato-Katz (KK) method, making it faster and more efficient for the visualisation of fertile eggs in stool samples. This modified KK method uses sodium acetate formalin as a fixative and reveals the intensity of infection in less than 1 h, reducing the diagnostic time without increasing the cost. This modified method may contribute to future epidemiological studies in both hospitals and the field due to its rapid and precise diagnostic, which allow for immediate treatment.

The influence of population density and food intake on the reproductive biology of Biomphalaria glabrata (Mollusca) and calcium proportion in snails experimentally infected with Schistosoma mansoni (Trematoda)

O presente trabalho teve por objetivo avaliar a influência da densidade populacional e da ingestão de alimento na sobrevivência e atividade reprodutiva de Biomphalaria glabrata não infectada e o efeito do carbonato de cálcio na emergência de cercárias de caramujos experimentalmente infectados, a fim de definir condições para maximizar a criação e a produção de cercárias em futuros estudos sobre esse modelo. Os resultados observados nesse estudo indicam que o aumento da densidade populacional tem efeito negativo sobre a sobrevivência e atividade reprodutiva de B. glabrata e aquantidade de alface fresca oferecida aos caramujos altera o número de ovos postos por molusco. Foi observada correlação significativa entre a quantidade de comida ingerida por dia e o número de ovos produzidos por molusco, bem como o número de massas ovígeras e ovos por massa ovígera.Além disso, a sobrevivência dos caramujos infectados foi diretamente associada a quantidade de carbonato de cálcio e a emergência de cercárias foi inversamente proporcional a quantidade de carbonato de cálcio. Esse estudo auxilia na compreensão da influência da densidade populacional e a ingestão de alimento na biologia reprodutiva de moluscos mantidos em colônias. Em relação à emergência de cercárias, suplementos de cálcio não devem ser adicionados nos criadouros decaramujos infectados com Schistosoma mansoni, tendo em vista que essa ação diminui a quantidade de cercárias eliminadas por caramujo.

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.

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.