A promising new target to control fasciolosis: Fasciola hepatica leucine aminopeptidase 2.

Parasite M17 leucine aminopeptidases (LAPs) have been associated with critical roles in different key functions such as the nutrition, migration, and invasion of the natural host. Native or recombinant LAP used as a vaccine antigen has proved effective to elicit protection against Fasciola hepatica infection in sheep, pointing to potential vaccine candidates against fascioliasis in ruminant species. Previously, the FhLAP1, abundantly secreted in vitro by the mature adult parasite was used as a vaccine antigen obtaining promising protection results against F. hepatica challenge in small ruminants. Here, we report the biochemical characterization of a second recombinant LAP (FhLAP2) associated with the juvenile stage of F. hepatica. FhLAP2 showed aminopeptidase activity using different synthetic substrates, including leucine, arginine, and methionine and was increased in the presence of Mn+ 2 and Mg+ 2. The activity was inhibited by bestatin, 1,10-phenanthroline, and EDTA, specific inhibitors of aminopeptidase and/or metalloproteases. Finally, the recombinant FhLAP2 functional form was tested in combination with Freund's incomplete adjuvant in an immunization trial in mice followed by an experimental challenge with F. hepatica metacercariae. The immunization with FhLAP2/FIA resulted in a significant reduction of parasite recovery compared to control groups. The immunized group elicited total specific IgG and subclasses IgG1 and IgG2 antibody responses. This study highlights the potential of a new candidate vaccine formulation with potential applications in natural ruminant hosts, especially those targeting the juvenile stage.

Fasciola gigantica vaccine construct: an in silico approach towards identification and design of a multi-epitope subunit vaccine using calcium binding EF-hand proteins.

Continuous attempts have been made to pinpoint candidate vaccine molecules and evaluate their effectiveness in order to commercialise such vaccines for the treatment of tropical fascioliasis in livestock. The pathophysiology of fascioliasis can be related to liver damage brought on by immature flukes that migrate and feed, as well as immunological reactions to chemicals produced by the parasites and alarm signals brought on by tissue damage. Future research should, in our opinion, concentrate on the biology of invasive parasites and the resulting immune responses, particularly in the early stages of infection. The goal of the current study was to use the calcium-binding proteins from F. gigantica to create a multi-epitope subunit vaccine. The adjuvant, B-cell epitopes, CTL epitopes, and HTL epitopes that make up the vaccine construct are all connected by certain linkers. The antigenicity, allergenicity, and physiochemical properties of the vaccine construct were examined. The vaccine construct was docked with toll-like receptor 2, and simulations of the molecular dynamics of the complex's stability, interaction, and dynamics were run. After performing in silico cloning and immunosimulation, it was discovered that the construct was suitable for further investigation. New vaccination technologies and adjuvant development are advancing our food safety procedures since vaccines are seen as safe and are accepted by the user community. This research is also applicable to the F. hepatica system.

Assessment of the molluscicidal impact of extracted chlorophyllin on some biochemical parameters in the nervous tissue and histological changes in Biomphalaria alexandrina and Lymnaea natalensis snails.

Biomphalaria alexandrina and Lymnaea natalensis snails are the intermediate hosts of schistosomiasis and fasciolosis. The aim of the present study is to evaluate the molluscicidal activity of chlorophyll extract as a photodynamic substance against these snails and how it affected its tissues and the biological system. Chlorophyllin was extracted from deep-frozen Moringa oleifera leaves, and then it was transformed into water-soluble chlorophyllin. The present results showed that it had a molluscicidal activity on B. alexandrina snails (LC50 17.6 mg/l; LC90 20.9 mg/l) and L. natalensis snails (LC50 4.3 mg/l; LC90 6.8 mg/l). Exposing B. alexandrina snails to the sublethal concentrations (LC0, LC10, and LC25) resulted in a significant reduction in their survival rates. Regarding its effect on biochemical parameters, chlorophyllin significantly reduced the acetylcholinesterase activity, protein content, and alkaline and acid phosphatase activity in B. alexandrina nervous tissue compared to the control group. Histopathological changes occurred in the digestive gland of treated B. alexandrina snails where cells lost their nuclei, vacuolated, degenerated, and ruptured, and the lumen increased. Photosynthesizing materials like chlorophyllin are new approaches to control schistosomiasis and fasciolosis in developing countries by affecting their intermediate host. These materials were cheap and environmentally safe to replace the synthetic molluscicides for snail control.

Moderate protection is induced by a chimeric protein composed of leucine aminopeptidase and cathepsin L1 against Fasciola hepatica challenge in sheep.

Leucine aminopeptidase (FhLAP) and cathepsin L1 (FhCL1) of Fasciola hepatica play a critical role in parasite feeding, migration through host tissue, and immune evasion. These antigens have been tested for immune protection as single components with variable degrees of success. The chimeric-protein approach could improve protection levels against fasciolosis. Previously, we reported the design and construction of a chimeric protein composed of antigenic sequences of FhLAP and FhCL1 of F. hepatica. The goal of the present study was to express and evaluate the immune-protective capacity of this chimeric protein (rFhLAP-CL1) in sheep. Animals were randomly allocated into five groups with five animals in each group. Groups 1, 2 and 3 were immunized twice with 100 µg, 200 µg and 400 µg of rFhLAP-CL1 emulsified with Quil A adjuvant, whereas groups 4 and 5 were the adjuvant control and infection control groups, respectively. The animals were then challenged with 200 metacercariae two weeks after the rFhLAP-CL1 booster. The fluke burden was reduced by 25.5%, 30.7% (p < 0.05) and 46.5% (p < 0.01) in sheep immunized with 100 µg, 200 µg and 400 µg of chimeric protein, respectively, in comparison to the infection control group. There was a reduction of 22.7% (p < 0.05) and 24.4% (p < 0.01) in fecal egg count in groups 2 and 3, respectively, compared to the infection control group. Sheep immunized with chimeric protein produced F. hepatica excretion-secretion product-specific total IgG antibody, which were increased after challenge. Moreover, the levels of rFhLAP-CL1-specific IgG1 and IgG2 isotypes in immunized sheep increased rapidly two weeks after the first immunization and were significantly more elevated than those of the control groups, indicating a mixed Th1/Th2 response. This is a preliminary evaluation of the chimeric protein rFhLAP-CL1 as a possible immunogen against F. hepatica infection in sheep.

The Battle of Worldviews: A Case Study of Liver Fluke Infection in Khon Kaen, Thailand.

Control efforts to reduce infection from the parasitic flatworm Opisthorchis viverrini have progressed through understanding the epidemiology of Opisthorchis viverrini, antiparasitic drug developments, technological innovations, health education promoting cooking of fish, and improved hygienic defecation. Yet the problem persists. The case study method was used to examine the fundamental cause of the liver fluke infection problem. Evidence shows that the liver fluke-infected population does not care about living a long life. For them, suffering and death are simply a part of life, and expected. Thus, the cause(s) leading to death is not important. They believe morally bad actions, and predetermined fate associated with kamma in Buddhism, play a big role whether or not one is infected with the liver fluke. Health interventions may be made more effective if they take into account the liver fluke-infected population's worldviews about ethics, morality, life, and death. We researchers should not feel concerned only about medically determined causes of death.

Transcriptome profiling of gene expression during immunisation trial against Fasciola hepatica: identification of genes and pathways involved in conferring immunoprotection in a murine model.

BACKGROUND: Fasciolosis remains a significant food-borne trematode disease causing high morbidity around the world and affecting grazing animals and humans. A deeper understanding concerning the molecular mechanisms by which Fasciola hepatica infection occurs, as well as the molecular basis involved in acquiring protection is extremely important when designing and selecting new vaccine candidates. The present study provides a first report of microarray-based technology for describing changes in the splenic gene expression profile for mice immunised with a highly effective, protection-inducing, multi-epitope, subunit-based, chemically-synthesised vaccine candidate against F. hepatica. METHODS: The mice were immunised with synthetic peptides containing B- and T-cell epitopes, which are derived from F. hepatica cathepsin B and amoebapore proteins, as novel vaccine candidates against F. hepatica formulated in an adjuvant adaptation vaccination system; they were experimentally challenged with F. hepatica metacercariae. Spleen RNA from mice immunised with the highest protection-inducing synthetic peptides was isolated, amplified and labelled using Affymetrix standardised protocols. Data was then background corrected, normalised and the expression signal was calculated. The Ingenuity Pathway Analysis tool was then used for analysing differentially expressed gene identifiers for annotating bio-functions and constructing and visualising molecular interaction networks. RESULTS: Mice immunised with a combination of three peptides containing T-cell epitopes induced high protection against experimental challenge according to survival rates and hepatic damage scores. It also induced differential expression of 820 genes, 168 genes being up-regulated and 652 genes being down-regulated, p value <0.05, fold change ranging from -2.944 to 7.632. A functional study of these genes revealed changes in the pathways related to nitric oxide and reactive oxygen species production, Interleukin-12 signalling and production in macrophages and Interleukin-8 signalling with up-regulation of S100 calcium-binding protein A8, Matrix metallopeptidase 9 and CXC chemokine receptor 2 genes. CONCLUSION: The data obtained in the present study provided us with a more comprehensive overview concerning the possible molecular pathways implied in inducing protection against F. hepatica in a murine model, which could be useful for evaluating future vaccine candidates.

In vitro phytotherapy of vector snails by binary combinations of larvicidal active components in effective control of fascioliasis.

A food-borne trematode infection fascioliasis is one among common public health problems worldwide. It caused a great economic loss for the human race. Control of snail population below a certain threshold level is one of the important methods in the campaign to reduce the incidence of fascioliasis. The life cycle of the parasite can be interrupted by killing the snail or Fasciola larva redia and cercaria inside of the snail Lymnaea acuminata. In vitro toxicity of different binary combinations (1:1 ratio) of plant-derived larvicidal active components such as citral, ferulic acid, umbelliferone, azadirachtin and allicin against Fasciola redia and cercaria were tested. The mortality of larvae was observed at 2h, 4h, 6h and 8h of treatment. In in vitro condition azadirachtin + allicin (1:1 ratio) was highly toxic against redia and cercaria (8h LC50 0.006 and 0.005 mg/L). Toxicity of citral + ferulic acid was lowest against redia and cercaria larvae.

In vitro PHYTOTHERAPY OF VECTOR SNAILS BY BINARY COMBINATIONS OF LARVICIDAL ACTIVE COMPONENTS IN EFFECTIVE CONTROL OF FASCIOLIASIS / Fitoterapia in vitro de caramujos vetores por combinações binárias de componentes ativos larvicidas como controle efetivo de fasciolíase

SUMMARY A food-borne trematode infection fascioliasis is one among common public health problems worldwide. It caused a great economic loss for the human race. Control of snail population below a certain threshold level is one of the important methods in the campaign to reduce the incidence of fascioliasis. The life cycle of the parasite can be interrupted by killing the snail or Fasciola larva redia and cercaria inside of the snail Lymnaea acuminata. In vitro toxicity of different binary combinations (1:1 ratio) of plant-derived larvicidal active components such as citral, ferulic acid, umbelliferone, azadirachtin and allicin against Fasciola redia and cercaria were tested. The mortality of larvae was observed at 2h, 4h, 6h and 8h of treatment. In in vitro condition azadirachtin + allicin (1:1 ratio) was highly toxic against redia and cercaria (8h LC50 0.006 and 0.005 mg/L). Toxicity of citral + ferulic acid was lowest against redia and cercaria larvae. .

RESUMO A infecção alimentar pelo trematóide da fasciolíase é uma dentre os mais comuns problemas de saúde pública mundiais, causando grande prejuízo econômico para a humanidade. Controle da população de caramujos abaixo de determinado nível é um dos métodos no campo mais importantes para a redução da incidência da fasciolíase. O ciclo de vida do parasita pode ser interrompido pela morte do caramujo ou da larva redia e cercária da Fasciola dentro da Lymnaea acuminata. Foi testada a toxicidade in vitro das diferentes combinações binárias (relação 1:1) entre os vários componentes larvicidas ativos da planta tais como citral, ácido ferúlico, umbeliferone, azadiractina, e alicina contra a Fasciola redia e a cercária. A mortalidade das larvas foi observada após duas, quatro, seis e oito horas de tratamento. A condição in vitro azadiractina + alicina (relação 1:1) foi altamente tóxica contra redia e cercária (8h LC50 0,006 e 0,005 mg/L). Toxicidade do citral + ácido ferúlico foi a mais baixa contra redia e larvas de cercária. .

Immunization with recombinant leucine aminopeptidase showed protection against Fasciola gigantica in mice.

Leucine aminopeptidase (LAP) is expressed in all stages of Fasciola gigantica and, hence, is considered as a potential vaccine candidate. In this study, we have tested a vaccine potential of LAP and the types of immune responses it elicited in vaccinated mice. Recombinant F. gigantica leucine aminopeptidase (rFgLAP) was expressed in Escherichia coli, BL21 (DE3). The imprinting control region mice subcutaneously immunized with 50 µg of rFgLAP combined with Freund's adjuvant (n = 10) exhibited a significant reduction in worm recoveries when compared with non-immunized and Freund's adjuvant controls at 60.8 and 64.3%, respectively, and both T helper (Th)1 and Th2 humoral immune responses were elicited in the hosts as reflected by the levels of IgG1 and IgG2a, with Th2 predominating. The levels of IgG1- and IgG2a-specific antibodies to rFgLAP were inversely and significantly correlated with the numbers of worm recoveries. The rFgLAP-vaccinated mice showed significantly reduced levels of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase and liver damage. These indicated that rFgLAP has a potential as a vaccine candidate against F. gigantica, whose efficacy will be studied further in economic animals including cattle, sheep, and goat.

Pochonia chlamydosporia in the biological control of Fasciola hepatica in cattle in Southeastern Brazil.

Biological control with the use of nematophagous fungi has been described very successfully by many authors and presents itself as a complementary control method, acting on the free-living forms of helminths. The efficacy of a formulation containing the fungus Pochonia chlamydosporia in controlling Fasciola hepatica eggs in faeces was evaluated in an experimental field assay. Two bovine groups (six animals each) were used: A (control) and B (treated with fungus). At 30 days after deworming, the animals were separated into two similar paddocks with flooded areas and were given pellets containing 25 % mycelial mass (group B) or no fungus (group A) at a dose of 1 g/10 kg body weight, twice a week, during 18 months. Faecal samples were harvested fortnightly in the animals of groups A and B and they were submitted at examination of quantitative sedimentation. The mean count of F. hepatica eggs per grams of faeces was significantly higher in group A (1.19) compared with those from group B (0.82) (P < 0.01). After 18 months, animals from group B had gained 42.33 kg above (17.82 % more by weight) (P < 0.01), compared with the control group (A). Every month, faecal samples from paddocks A and B were collected and they were incubated. P. chlamydosporia was identified only in sample source of the paddock B. It can be concluded that the application of this fungical formulation with P. chlamydosporia 25 % mycelial mass was effective in reducing the availability of eggs in the environment and reinfections in calves in natural conditions.

Vaccination of buffaloes with Fasciola gigantica recombinant glutathione S-transferase and fatty acid binding protein.

Fasciola gigantica, causative agent of tropical fasciolosis, inflicts substantial economic losses on the livestock industry, affecting severely buffalo productivity in the tropical countries. Very few vaccination trials with different target antigens against F. gigantica infection have been conducted in this host. Present study describes a vaccination trial in buffaloes with F. gigantica recombinant glutathione S-transferase and fatty acid binding protein. The two recombinant proteins were expressed in Escherichia coli and evaluated for their immunoprophylactic potential in buffalo calves, using montanide 70 M-VG, a mineral oil-based adjuvant, for delivering the antigens. Buffalo calves were distributed in three groups, with group I, II and III calves immunized with recombinant glutathione S-transferase, fatty acid binding protein and a cocktail of these two antigens, respectively. Immunization of the calves evoked a mixed IgG1 and IgG2 antibody response. Present vaccination trial in these animals achieved a maximum protection level of 35%, when the two antigens were used in combination. Eosinophils were measured in both immunized and non-immunized challenge control animals, which showed a steady increase in their count in response to immunization with both the antigens and infection with F. gigantica, respectively.

The recombinant gut-associated M17 leucine aminopeptidase in combination with different adjuvants confers a high level of protection against Fasciola hepatica infection in sheep.

Fasciola hepatica M17 leucine aminopeptidase (FhLAP) is thought to play a role in catabolizing peptides generated by the concerted activity of gut-associated endopeptidases on host polypeptides, thus releasing amino acids to be used in protein anabolism. In this study, a recombinant functional form of this homo hexameric metallopeptidase produced in Escherichia coli was used in combination with adjuvants of different types in a vaccination trial in Corriedale sheep against experimental challenge with F. hepatica metacercariae. The experimental assay consisted of 6 groups of 10 animals; 5 of the groups (1-5) were subcutaneously inoculated at weeks 0 and 4 with 100 µg of rFhLAP mixed with Freund's complete plus incomplete adjuvant (group 1), Alum (group 2), Adyuvac 50 (group 3), DEAE-D (group 4) and Ribi (group 5); the adjuvant control group (group 6) received Freund's adjuvant. Two weeks after the booster, the sheep were orally challenged with 200 metacercariae. Immunization with rFhLAP induced significant reduction in fluke burdens in all vaccinated groups: 83.8% in the Freund's group, 86.7% in the Alum group, 74.4% in the Adyuvac 50 group, 49.8% in the Ribi group and 49.5% in the DEAE-D group compared to the adjuvant control group. Morphometric analysis of recovered liver flukes showed no significant size modifications in the different vaccination groups. All vaccine preparations elicited specific IgG, IgG1 and IgG2 responses. This study shows that a liver fluke vaccine based on rFhLAP combined with different adjuvants significantly reduced worm burden in a ruminant species that was high in animals that received the enzyme along with the commercially approved adjuvants Alum and Adyuvac 50.

Molluscicidal activity of various solvent extracts from Solanum nigrum var. villosum L. aerial parts against Galba truncatula.

Molluscicidal activity of Solanum nigrum var. villosum (morelle velue) extracts and their fractions were tested against the mollusca gastropoda Galba truncatula intermediate host of Fasciola hepatica. The results indicated that the hydro-methanol (MeOH-H2O) immature fruit extract possess the highest molluscicidal activity (LC50 = 3.96 mg/L) against Galba truncatula compared with other tested compounds. After acido-basic treatment, the methanolic extract fraction isolated from the immature fruits and the richest in alkaloids was the most toxic (LC50 = 1.65 mg/L). The fractions richest in saponosides obtained from the hydromethanolic and methanolic extracts of immature fruits showed interesting molluscicidal activities (LC50 = 6.15 mg/L and LC50 = 7.91 mg/L, respectively). The observed molluscicide activity could be attributed to the presence of alkaloids or saponosides. So, the immature fruits of Solanum nigrum var. villosum could be substrates of choice for molluscicide activity. In addition, total alkaloids and saponosides present in this plant deserve further investigations in order to identify the active principles and demonstrate their activities on mollusks in their natural habitat. According to the World Health Organization's guidelines on screening for plant molluscicides, use of these fractions may add to the arsenal of methods to control snail transmitting fasciolosis in tropical and Third World countries where fasciolosis is a common disease.

Fasciola hepatica leucine aminopeptidase, a promising candidate for vaccination against ruminant fasciolosis.

Leucyl aminopeptidases (LAP) from different parasitic organisms are attracting attention as relevant players in parasite biology, and consequently being considered as candidates for drug and vaccine design. In fact, the highest protection level achieved in ruminant immunization by a native antigen was previously reported by us, using a purified LAP as immunogen in a sheep trial against fasciolosis. Here, we report the cloning of a full-length cDNA from adult F. hepatica encoding a member of the M17 family of LAP (FhLAP) and functional expression and characterization of the corresponding enzyme. FhLAP was closely related to Schistosoma LAPs, but interestingly distant from their mammalian host's homologues, and was expressed in all stages of the parasite life cycle. The recombinant enzyme, functionally expressed in Escherichia coli, showed a marked amidolytic preference against the synthetic aminopeptidase substrate l-leucine-7-amino-4-methylcoumarin (Leu-AMC) and was also active against Cys-AMC and Met-AMC. Both native and recombinant enzyme were stimulated by the addition of divalent cations predominantly Mn(2+), and strongly inhibited by bestatin and cysteine. Physico-chemical properties, localization by immunoelectron microscopy, MALDI-TOF analysis, and cross-reactivity of anti-rFhLAP immune serum demonstrated that the recombinant enzyme was identical to the previously purified gut-associated LAP from adult F. hepatica. Vaccination trials using rFhLAP for rabbit immunization showed a strong IgG response and a highly significant level of protection after experimental infection with F. hepatica metacercariae, confirming that FhLAP is a relevant candidate for vaccine development.

Progress in the development of Fasciola hepatica vaccine using recombinant fatty acid binding protein with the adjuvant adaptation system ADAD.

Fatty acid binding proteins (FABP) have been designed as a potential vaccine against fasciolosis. In this work, the immunoprophylaxis of the recombinant Fh15 FABP from F. hepatica (Fh15) in adjuvant/immunomodulator ADAD system was evaluated using mice and sheep challenged with F. hepatica. The ADAD system combines the Fh15 antigen with an immunomodulator (hydroalcoholic extract of Polypodium leucotomos; PAL) and/or an adjuvant (saponins of Quillaja saponaria; Qs) in a water/oil emulsion (30/70) with a non-mineral oil (Montanide). All the infected control mice died by 41-48 days post-infection. The mice vaccinated with ADAD only with PAL+Fh15 present a survival rate of 40-50% and those vaccinated with ADAD containing PAL+Qs+Fh15 had a survival rate of 50-62.5%. IgG1 antibodies were lower in surviving mice in comparison with non-surviving mice. The sheep vaccinated with ADAD PAL+Qs+Fh15 showed lower fluke recovery (43%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the ADAD system using recombinant fatty acid binding proteins from F. hepatica could be a good option to develop vaccines against F. hepatica.

Vaccination of mice and sheep with Fh12 FABP from Fasciola hepatica using the new adjuvant/immunomodulator system ADAD.

We evaluate the ability of a Fasciola hepatica FABP native antigen (Fh12) with a new vaccination system called ADAD to protect mice and sheep against an experimental F. hepatica infection. The vaccination protocol consists of a set of two injections. The first injection contains a micelle in which two components are included, saponin from Quillaja saponaria (Qs) and/or Anapsos (A) a Polypodium leucotomos hydroalcoholic extract, both emulsified in a non-mineral oil (Montanide) in a water/oil emulsion (30/70). This is subcutaneously injected to achieve the "adaptation" of the immune system to subsequent stimuli. The second injection contains in addition the Fh12 antigen. Two different experiments were carried out using two mouse strains (BALB/c and CD-1). Mice vaccinated with Qs+A+Fh12 presented a survival rate of 40%, when compared with control groups. Furthermore, we evaluated the efficiency of the vaccination in sheep against an experimental F. hepatica challenge. The vaccinated sheep presented lower fluke recovery (24.5%), number of eggs in bile fluid (58.1%) and faeces (40.3%) than control groups. The recovered flukes were shorter (32.7%), immature (34.0%) and with lower body mass (31.6%) than non-complete vaccinated sheep. Thus, the new ADAD system could be a good alternative for future vaccination experiments against fasciolosis.

Molluscicidal activity of Lawsonia inermis and its binary and tertiary combinations with other plant derived molluscicides.

Molluscicidal activity of leaf, bark and seed of Lawsonia inermis against Lymnaea acuminata and Indoplanorbis exustus was studied. Highest toxicity was observed in the seed of Lawsonia inermis. Toxicity of binary (1:1) and tertiary (1:1:1) combinations of the essential oil of cedar (Cedrus deodara Roxh) and neem (Azadirachta indica A. Juss), powder from bulb of garlic (Allium sativum Linn), and oleoresin extracted from rhizome of ginger (Zingiber officinale Rosc) with Lawsonia inermis and Embelia ribes fruit powder were studied against L. acuminata and I. exustus. L. inermis seed powder in combination with Cedrus deodara oil and Azadirachta indica oil was more toxic than their individual components and other combinations.

[Molluscacidal activity of Paraiso (Melia azedarach L.) (Meliaceae) on Lymnaea cubensis, host snail of fascioliasis]. / Actividad molusquicida del Paraiso (Melia azedarach L.) (Meliaceae) sobre Lymnaea cubensis, molusco vector de Fasciolosis.

INTRODUCTION: Fasciolosis is a source of serious economic loss in various regions of Brazil and when control measures of its host (molluscs) are not taken under favorable ecological conditions, isolated cases of human Fasciolosis may occur. Among the alternative measures for its control is the use of vegetable extracts and the purpose of this project is the assessment of the use of juice extracted from the fruit and seeds of the Paraiso plant (Melia azedarach L.) in the control of Lymnaea cubensis, the main vector of Fasciolosis in Cuba. MATERIAL AND METHOD: Various concentrations of the juice extracted from the Paraiso fruit (Melia azedarach L.) were tested to determine the average and maximum lethal doses (DL50 and DL90' respectively) used in a computerized PROBIT-LOG program. Seven experimental series were undertaken, 72 molluscs being used in each of them. Three groups of ten molluscs were tested to determine the effect on cardiac frequency, two of them being treated with CL50 = 0.88627 and CL90 = 1.7641, respectively, the third being used as control. RESULTS AND CONCLUSIONS: A considerable effect of both doses on the cardiac frequency of the mollusc studied was observed. The results were encouraging, for they demonstrated the potential use of this plant in the control of undesirable molluscs.

Actividad molusquicida del Paraiso (Melia azedarach L.) (Meliaceae) sobre Lymnaea cubensis, molusco vector de Fasciolosis / Molluscicidal activity of Paraiso (Melia azedarach L.) (Meliaceae) on Lymnaea cubensis, host snail of Fasciolosis

A Fasciolosis constitui em diversas regiöes fonte importante de perdas econômicas, e quando näo se adotam medidas de controle de seus hospedeiros (moluscos), junto a condiçöes ecológicas favoráveis, pode ocorrer o aparecimento de casos isolados de Fasciolosis humana. Dentro dos métodos alternativos para o seu controle está o uso de extratos vegetais e se tem pretendido avaliar o provável emprego do suco extraíso do fruto e sementes do Paraiso (Melia azedarach L.), no controle de Lymnaea cubensis, principal vetor da Fasciolosis em Cuba. Diferentes concentraçöes do suco extraído do fruto e semente do Paraiso (Melia azedarach L.) foram testadas para determinar as doses médias letais (DL50) e máxima (DL90) usando um programa computadorizado PROBIT-LOG. Sete séries experimentais foram realizadas, usando 72 moluscos em cada uma. Para determinar a influência sobre a freqüência cardíaca foram testados três grupos de 10 moluscos, dois foram tratados com as CL50=0,88627 e CL90=1,7641, respectivamente, enquanto que o terceiro foi considerado como controle. Observou-se uma marcada influência de ambas as doses sobre a freqüência cardíaca do molusco estudado. Os resultados foram alentadores, pois demonstraram um potencial uso dessa planta no controle de moluscos näo desejados