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OVICIDAL EFFECT OF PIPERACEAE SPECIES ON Biomphalaria glabrata, Schistosoma mansoni HOST

Efeito ovicida de espécies de Piperaceae em Biomphalaria glabrata, hospedeiro do Schistosoma mansoni

Abstracts

SUMMARY

Schistosomiasis is a neglected disease with public health importance in tropical and subtropical regions. An alternative to the disease control is the use of molluscicides to eliminate or reduce the intermediate host snail population causing a reduction of transmission in endemic regions. In this study nine extracts from eight Piperaceae species were evaluated against Biomphalaria glabrata embryos at blastula stage. The extracts were evaluated in concentrations ranging from 100 to 10 mg/L. Piper crassinervium and Piper tuberculatum extracts were the most active (100% of mortality at 20 mg/L and 30 mg/L respectively).

Schistosomiasis; Biomphalaria glabrata ; Embryos; Crude extracts; Piperaceae; Molluscicide


RESUMO

A esquistossomose é uma doença negligenciada de importância para a saúde pública em regiões tropicais e subtropicais. Uma alternativa para o controle da doença é o uso de moluscicidas para eliminar ou reduzir a população de caramujo hospedeiro, acarretando uma redução da transmissão da doença nas regiões endemicas. Neste estudo, nove extratos vegetais provenientes de oito espécies de Piperaceae foram expostos a embriões de Biomphalaria glabrata no estágio de blástula. Os extratos foram avaliados em concentrações que variaram entre 100 e 10 mg/L, sendo Piper crassinervium e Piper tuberculatum os extratos mais ativos (100% de mortalidade a 20 mg/L e 30 mg/L respectivamente).


INTRODUCTION

Schistosomiasis is one of the most prevalent, debilitating and neglected diseases of tropical and subtropical regions, such as Africa, Asia and South America. This disease is a relevant health and social-economic problem with more than 390-600 million people estimated to have been infected worldwide, while 800 million people remain under infection risk1111. Gray DJ, McManus DP, Li Y, Williams GM, Bergquist R, Ross AG. Schistosomiasis elimination: lessons from the past guide the future. Lancet Infect Dis. 2010;10:733-6. 2929. World Health Organization. Schistosomiasis: population requiring preventive chemotherapy and number of people treated in 2010. Wkly Epidemiol Rec. 2012;4:37-44..

Currently, the main strategy to control schistosomiasis is based on the periodic treatment of people living in risk areas with anti-schistosomicidal drugs in order to reduce morbidity and transmission2828. World Health Organization. Working to overcome the global impact of neglected tropical diseases. First WHO report on neglected tropical diseases. Geneva: WHO; 2010. [WHO/HTM/NTD/2010.1].. However, evidence indicates that resistance and tolerance to praziquantel, a main drug used in Schistosoma mansoni treatment, have been increasing66. Cioli D. Praziquantel: is there real resistance and are there alternatives? Curr Opin Infect Dis. 2000;13:659-63. 99. Doenhoff MJ, Cioli D, Utzinger J. Praziquantel: mechanisms of action, resistance and new derivatives for schistosomiasis. Curr Opin Infect Dis. 2008;21:659-67..

Freshwater snails of Biomphalaria genus play a major role as intermediate hosts in the transmission of S. mansoni because an intense multiplication of parasites occurs in these snails. Thus, any strategy to control snail populations for reduction of schistosomiasis transmission in endemic regions should consider some treatment at this critical stage1515. Lardans VD, Dissous C. Snail control strategies for reduction of schistosomiasis transmission. Parasitol Today. 1998;14:413-7..

Currently, niclosamide marketed as Bayluscide® is the most important molluscicide recommended by World Health Organization (WHO) to control the host of schistosomiasis3232. World Health Organization. The role of mollusciciding in schistosomiasis control. Geneva: WHO; 1992. [WHO/SCHISTO/92.107;1992].. However, niclosamide, has plenty of adverse factors, such as, non-selective toxicity, that harms other aquatic organisms; high cost; instability in sunlight, which in addition requires frequent reapplication due to the permanence of surviving snails after its application11. Abreu FC, Goulart MOF, Brett AMO. Detection of the damage caused to DNA by niclosamide using an electrochemical DNA-biosensor. Biosens Bioelectron. 2002;17:913-9. 1010. Giovanelli A, Silva CL, Medeiros L, Vasconcellos MC. The molluscicidal activity of niclosamide (Bayluscide WP70®) on Melanoides tuberculata (Thiaridae), a snail associated with habitats of Biomphalaria glabrata (Planorbidae). Mem Inst Oswaldo Cruz. 2002;97:743-5. 3232. World Health Organization. The role of mollusciciding in schistosomiasis control. Geneva: WHO; 1992. [WHO/SCHISTO/92.107;1992].. Thus, novel natural molluscicidal should be investigated as a possible alternative to synthetic products1616. Luna JS, Santos AF, Lima MRF, Omena MC, Mendonça FAC, Bieber LW, et al. A study of the larvicidal and molluscicidal activities of some medicinal plants from northeast Brazil. J Ethnopharmacol. 2005;97:199-206. 1717. Mello-Silva CC, Vilar MM, Bezerra JCB, Vasconcellos MC, Pinheiro J, Rodrigues ML. Reproductive activity alterations on the Biomphalaria glabrata exposed to Euphorbia splendens var. hislopii latex. Mem Inst Oswaldo Cruz. 2007;102:671-4. 1818. Miyasato PA, Kawano T, Freitas JC, Berlinck RGS, Nakano E, Tallarico LF. Molluscicidal activity of some marine substances against the snail Biomphalaria glabrata (Mollusca, Planorbidae). Parasitol Res. 2012;110:1873-9. 2525. Rapado LN, Nakano E, Ohlweiler FP, Kato MJ, Yamaguchi LF, Pereira CA, et al. Molluscicidal and ovicidal activities of plant extracts of the Piperaceae on Biomphalaria glabrata (Say, 1818). J Helminthol. 2011;85:66-72..

The genus Piper belongs to the Piperaceae family and includes more than 2000 species widely distributed in the tropical and subtropical regions of the world1313. Jaramillo MA, Manos PS, Zimmer EA. Phylogenetic relationships of the perianthless Piperales: reconstructing the evolution of floral development. Int J Plant Sci. 2004;165:403-16.. Piper species are important plants used in Chinese medicine, in the Indian Ayurvedic system and folk medicine practices of Latin America and West Indies. Furthermore, studies with plants from the Piperaceae family have shown a great diversity of secondary metabolites with biological activity1414. Kato M, Furlan M. Chemistry and evolution of the Piperaceae. Pure Appl Chem. 2007;79:529-38..

Recently, schistosomicidal and molluscicidal activities of Piperaceae species have been described and this family is considered promising for studies of schistosomiasis prevention and control1919. Moraes J, Nascimento C, Lopes PO, Nakano E, Yamaguchi LF, Kato MJ, et al. Schistosoma mansoni: in vitro schistosomicidal activity of piplartine. Exp Parasitol. 2011;127:357-64. 2020. Moraes J, Nascimento C, Yamaguchi LF, Kato MJ, Nakano E. Schistosoma mansoni: in vitro schistosomicidal activity and tegumental alterations induced by piplartine on schistosomula. Exp Parasitol. 2012;132:222-7. 2525. Rapado LN, Nakano E, Ohlweiler FP, Kato MJ, Yamaguchi LF, Pereira CA, et al. Molluscicidal and ovicidal activities of plant extracts of the Piperaceae on Biomphalaria glabrata (Say, 1818). J Helminthol. 2011;85:66-72..

In this work, nine crude extracts of eight Piperaceae species were assessed for ovicidal activity against Biomphalaria glabrata (Say, 1818) embryos.

MATERIALS AND METHODS

Ovicidal assay was performed according to the methodology recommended by the WHO3030. World Health Organization. Molluscicide screening and evaluation. Bull World Health Organ. 1965;334:567-81. 3131. World Health Organization. Report of the scientific working group on plant molluscicides. Bull World Health Organ. 1983;12:1-11. and the experimental procedures were according to the accepted principles of animal welfare in experimental science.

Embryos were obtained from Biomphalaria glabrata snail originally from Belo Horizonte, MG, Brazil, and kept in a laboratory under light, temperature and feeding controlled.

Plant material: Fresh material of each Piperaceae species (Table 1) was collected by Dr Massuo Jorge Kato from Universidade de São Paulo (Chemical Institute) and identified by Dr Elsie F. Guimarães from Instituto de Pesquisas Jardim Botânico do Rio de Janeiro. Vouchers were deposited in the Herbarium of Jardim Botânico do Rio de Janeiro, Brazil.

Table 1
Piperaceae species screened for ovicidal activity in B. glabrata embryos

Preparation of extracts and samples: Selected parts of freshly collected plant material were separated, immediately air dried and finally dried in an oven at 45 °C for 24 h (Table 1). Material was ground and extracted with ethyl acetate: methanol (2:1) at room temperature (25-28 °C) three times and filtered. Extracts were concentrated to dryness under vacuum in a rotary evaporator and stored at -20 °C.

A stock solution containing 1 g/L of each extract was prepared by suspending 10 mg of extract in 0.1 mL of 99.9% dimethylsulphoxide (DMSO: Aldrich, Milwaukee, WI, USA) and making up to 10 mL with dechlorinated water. Stock solutions were diluted with dechlorinated water in order to provide assay solutions.

Assay for ovicidal activity: Egg masses with embryos at blastula stage55. Camey T, Verdonk NH. The early development of the snail Biomphalaria glabrata (Say) and the origin of the head organs. Netherlands J Zool. 1969;20:93-121. were exposed to Piperaceae extracts (Table 1) firstly at 100 mg/L for 24 hours and observed for seven days. Inactive extracts at this concentration were not further investigated since crude preparation of plant material should be active at 100 mg/L or less, according to WHO3131. World Health Organization. Report of the scientific working group on plant molluscicides. Bull World Health Organ. 1983;12:1-11..

Tests were carried out with egg masses laid on small pieces of plastic sheet that had been left floating on the aquaria water. The pieces of plastic sheets with adhered egg masses were carefully transferred to Petri dishes, where they were further exposed to testing solutions. For each concentration, five egg masses were used and assays were repeated three times. The number of snail embryos to each concentration is indicated in Tables 2 and 3. At the end of exposure (24 hours), egg masses were washed with dechlorinated water. Petri dishes containing egg masses were kept within climatic chambers under controlled temperature (25 °C ± 1 °C). All egg masses were examined daily under a stereomicroscope up to the 7th day. Embryos were considered as dead whenever disintegrating embryonic forms were noted within the egg and or, at later developmental stages and no heartbeats were detected. A negative control group was maintained in 1% DMSO on dechlorinated water under the same experimental conditions. Bayluscide WP70® was used as a positive control22. Andrews P, Thyssen J & Lorke D. The biology and toxicology of molluscicides, Bayluscide. Pharmacol Ther. 1983;19:245-95..

Table 2
Ovicidal effect of Piperaceae extracts at blastula stage at 100 mg/L
Table 3
Species with ovicidal activity at the blastula stage in concentrations lower than 100 mg/L

The LC50 (50% lethal concentration) and the 95% confidence intervals for active extracts were estimated using Trimmed Spearman-Karber Method1212. Hamilton MA, Russo RC, Thurston RV. Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays. Environ Sci Technol. 1977;11:714-8..

RESULTS AND DISCUSSION

Three extracts from Piperaceae species were active with 100% of embryo mortality at 100 mg/L: P. crassinervium inflorescence extract and P. tuberculatum inflorescence and leaf extracts (Table 2). Both species showed 100% of lethality at 100 mg/L during the 24 h exposure period (Table 2, Fig. 1). These extracts were also evaluated at lower concentrations and inflorescence extract of P. crassinervium was more active than P. tuberculatum inflorescence and leaf extracts (100% of mortality at 20 mg/L and 30 mg/L respectively) (Table 3).

Fig. 1
Embryos of B. glabrata at blastula stage during the exposure period. A- Dead embryos exposure to leaf extract of P. tuberculatum at 100 mg/L and B- Normal embryo (control group).

No increase of embryolethality was observed in embryos exposed to leaf extracts of P. solmsianum, P. callosum, P.oreophylla, P. tretraphylla, P. mallacophyllum and P. glabella at 100 mg/L (Table 2). Percentage of dead embryos in control groups during all the study was not higher than 1.2%.

RAPADO et al. (2011) evaluated the molluscicidal effect of P. crassinervium leaf extract in B. glabrata adult and embryos at blastula stage obtained 100% of mortality at 60 mg/L and 50 mg/L respectively. This species has flavonoids and prenylated benzoic acid as major compound in leaf, classes of compounds with molluscicide activities already described88. Danelutte AP, Lago JHG, Young MCM, Kato MJ. Antifungal flavanones and prenylated hydroquinones from Piper crassinervium Kunt. Phytochemistry. 2003;64:555-9. 2424. Orjala J, Erdelmeier CAJ, Wright AD, Rali T, Sticher O. Five new prenylated p-hydroxybenzoic acid-derivatives with antimicrobial and molluscicidal activity from Piper aduncum leaves. Planta Med. 1993;59:546-51.. Nevertheless is not known if those compounds are responsible for the molluscicidal activity obtained in this study using inflorescence extract.

The P. tuberculatum is largely used in folk medicine as a sedative and antidote for snake bite. It has been shown that extracts and amides isolated from P. tuberculatum fruit and seeds have also a potent antifungal activity against Cladosporium sphaerospermum (100% active in 5 µg) and parasitic activity in Trypanosoma cruzi (IC50 = 17.2 µg/mL in epimastigote), Leishmania donovani (IC50 = 7.5 µg/mL in promastigote) and S. mansoni (100% mortality in 9.5 µM)44. Bodiwala H, Singh G, Singh R, Dey CS, Sharma SS, Bhutani KK, et al. Antileishmanial amides and lignans from Piper cubeba and Piper retrofractum. J Nat Med. 2007;61:418-21. 77. Cotinguiba F, Regasini LO, Bolzani VS, Debonsi HM, Passerini GD, Cicarelli RMB, et al. Piperamides and their derivatives as potential anti-trypanosomal agents. Med Chem Res. 2009;18:703-11. 1919. Moraes J, Nascimento C, Lopes PO, Nakano E, Yamaguchi LF, Kato MJ, et al. Schistosoma mansoni: in vitro schistosomicidal activity of piplartine. Exp Parasitol. 2011;127:357-64. 2121. Navickiene HMD, Alécio AC, Kato MJ, Bolzani VS, Young MC, Cavalheiro AJ, et al. Antifungal amides from Piper hispidum and Piper tuberculatum. Phytochemistry. 2000;55:621-6 2222. Navickiene HMD, Bolzani VS, Kato MJ, Pereira AM, Bertoni BW, França SC, Furlan M. Quantitative determination of anti-fungal and insecticide amides in adult plants, plantlets and callus from Piper tuberculatum by reverse-phase high-performance liquid chromatography. Phytochemical Anal. 2003;14:281-4. 2727. Silva RV, Navickiene HM, Kato MJ, Bolzani VS, Méda CI, Young MC, et al. Antifungal amides from Piper arboreum and Piper tuberculatum. Phytochemistry. 2002;59:521-7.. In this study, leaves and inflorescences extract showed ovicidal activity in equal concentrations, suggesting the possible presence of active compounds in both parts of the plant.

Studies with molluscicides compounds show that it is usual to obtain the death of B. glabrata snail but not the embryos1818. Miyasato PA, Kawano T, Freitas JC, Berlinck RGS, Nakano E, Tallarico LF. Molluscicidal activity of some marine substances against the snail Biomphalaria glabrata (Mollusca, Planorbidae). Parasitol Res. 2012;110:1873-9. 2323. Oliveira-Filho EC, Geraldino BR, Coelho DR, De-Carvalho RR, Paumgartten FJ. Comparative toxicity of Euphorbia milii latex and synthetic molluscicides to Biomphalaria glabrata embryos. Chemosphere. 2010;81:218-27. 2525. Rapado LN, Nakano E, Ohlweiler FP, Kato MJ, Yamaguchi LF, Pereira CA, et al. Molluscicidal and ovicidal activities of plant extracts of the Piperaceae on Biomphalaria glabrata (Say, 1818). J Helminthol. 2011;85:66-72.. The lack of ovicidal activity allows the permanence of the snail host in the environment, maintaining the transmission of schistosomiasis.

The Euphorbiaceae species are known for producing latex with molluscicidal activity restricted to B. glabrata adults (100% mortality at 1.5 mg/L)33. Baptista DF, Vasconcellos MC, Lopes FE, Silva IP, Schall VT. Perspectives of using Euphorbia splendens as a molluscicide in schistosomiasis control programs. Southeast Asian J Trop Med Public Health. 1994;25:419-24. 2626. Schall VT, Vasconcellos MC, Souza CP, Baptista DF. The molluscicidal activity of crown of christ (Euphorbia splendens var. hislopii) latex on snails acting as intermediate hosts of Schistosoma mansoni and Schistosoma haematobium. Am J Trop Med Hyg. 1998;58:7-10.. Different from this, Piper species are lethal to B. glabrata adults and embryos in concentrations recommended by WHO as Piper cuyabanum (100% lethal for adults and embryos at 20 mg/L), Piper aduncum (100% lethal in adults at 10 mg/L and embryos at 50 mg/L) and Piper hostmannianum (100% lethal in adults at 40 mg/L and embryos at 20 mg/L)2525. Rapado LN, Nakano E, Ohlweiler FP, Kato MJ, Yamaguchi LF, Pereira CA, et al. Molluscicidal and ovicidal activities of plant extracts of the Piperaceae on Biomphalaria glabrata (Say, 1818). J Helminthol. 2011;85:66-72..

In this work, three extracts from two Piperaceae species were lethal to B. glabrata embryos under concentrations recommended by WHO3131. World Health Organization. Report of the scientific working group on plant molluscicides. Bull World Health Organ. 1983;12:1-11.. Thus P. tuberculatum and P. crassinervium extracts were active at 30 mg/L and 20 mg/L respectively which make them species targets for isolation and identification of ovicidal compounds since these species are also active in B. glabrata adult2525. Rapado LN, Nakano E, Ohlweiler FP, Kato MJ, Yamaguchi LF, Pereira CA, et al. Molluscicidal and ovicidal activities of plant extracts of the Piperaceae on Biomphalaria glabrata (Say, 1818). J Helminthol. 2011;85:66-72..

This article is dedicated to the memory of Dr. Toshie Kawano, a researcher who initially coordinated this work and devoted her studies to schistosomiasis control.

This work was supported by grants from FAPESP (Project number 09/51850-9) coordinated by Prof. Dr Massuo Jorge Kato.

The authors are grateful to Dr Massuo Jorge Kato for providing access to the Piperaceae extracts.

REFERENCES

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    Abreu FC, Goulart MOF, Brett AMO. Detection of the damage caused to DNA by niclosamide using an electrochemical DNA-biosensor. Biosens Bioelectron. 2002;17:913-9.
  • 2
    Andrews P, Thyssen J & Lorke D. The biology and toxicology of molluscicides, Bayluscide. Pharmacol Ther. 1983;19:245-95.
  • 3
    Baptista DF, Vasconcellos MC, Lopes FE, Silva IP, Schall VT. Perspectives of using Euphorbia splendens as a molluscicide in schistosomiasis control programs. Southeast Asian J Trop Med Public Health. 1994;25:419-24.
  • 4
    Bodiwala H, Singh G, Singh R, Dey CS, Sharma SS, Bhutani KK, et al. Antileishmanial amides and lignans from Piper cubeba and Piper retrofractum. J Nat Med. 2007;61:418-21.
  • 5
    Camey T, Verdonk NH. The early development of the snail Biomphalaria glabrata (Say) and the origin of the head organs. Netherlands J Zool. 1969;20:93-121.
  • 6
    Cioli D. Praziquantel: is there real resistance and are there alternatives? Curr Opin Infect Dis. 2000;13:659-63.
  • 7
    Cotinguiba F, Regasini LO, Bolzani VS, Debonsi HM, Passerini GD, Cicarelli RMB, et al. Piperamides and their derivatives as potential anti-trypanosomal agents. Med Chem Res. 2009;18:703-11.
  • 8
    Danelutte AP, Lago JHG, Young MCM, Kato MJ. Antifungal flavanones and prenylated hydroquinones from Piper crassinervium Kunt. Phytochemistry. 2003;64:555-9.
  • 9
    Doenhoff MJ, Cioli D, Utzinger J. Praziquantel: mechanisms of action, resistance and new derivatives for schistosomiasis. Curr Opin Infect Dis. 2008;21:659-67.
  • 10
    Giovanelli A, Silva CL, Medeiros L, Vasconcellos MC. The molluscicidal activity of niclosamide (Bayluscide WP70®) on Melanoides tuberculata (Thiaridae), a snail associated with habitats of Biomphalaria glabrata (Planorbidae). Mem Inst Oswaldo Cruz. 2002;97:743-5.
  • 11
    Gray DJ, McManus DP, Li Y, Williams GM, Bergquist R, Ross AG. Schistosomiasis elimination: lessons from the past guide the future. Lancet Infect Dis. 2010;10:733-6.
  • 12
    Hamilton MA, Russo RC, Thurston RV. Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays. Environ Sci Technol. 1977;11:714-8.
  • 13
    Jaramillo MA, Manos PS, Zimmer EA. Phylogenetic relationships of the perianthless Piperales: reconstructing the evolution of floral development. Int J Plant Sci. 2004;165:403-16.
  • 14
    Kato M, Furlan M. Chemistry and evolution of the Piperaceae. Pure Appl Chem. 2007;79:529-38.
  • 15
    Lardans VD, Dissous C. Snail control strategies for reduction of schistosomiasis transmission. Parasitol Today. 1998;14:413-7.
  • 16
    Luna JS, Santos AF, Lima MRF, Omena MC, Mendonça FAC, Bieber LW, et al. A study of the larvicidal and molluscicidal activities of some medicinal plants from northeast Brazil. J Ethnopharmacol. 2005;97:199-206.
  • 17
    Mello-Silva CC, Vilar MM, Bezerra JCB, Vasconcellos MC, Pinheiro J, Rodrigues ML. Reproductive activity alterations on the Biomphalaria glabrata exposed to Euphorbia splendens var. hislopii latex. Mem Inst Oswaldo Cruz. 2007;102:671-4.
  • 18
    Miyasato PA, Kawano T, Freitas JC, Berlinck RGS, Nakano E, Tallarico LF. Molluscicidal activity of some marine substances against the snail Biomphalaria glabrata (Mollusca, Planorbidae). Parasitol Res. 2012;110:1873-9.
  • 19
    Moraes J, Nascimento C, Lopes PO, Nakano E, Yamaguchi LF, Kato MJ, et al. Schistosoma mansoni: in vitro schistosomicidal activity of piplartine. Exp Parasitol. 2011;127:357-64.
  • 20
    Moraes J, Nascimento C, Yamaguchi LF, Kato MJ, Nakano E. Schistosoma mansoni: in vitro schistosomicidal activity and tegumental alterations induced by piplartine on schistosomula. Exp Parasitol. 2012;132:222-7.
  • 21
    Navickiene HMD, Alécio AC, Kato MJ, Bolzani VS, Young MC, Cavalheiro AJ, et al. Antifungal amides from Piper hispidum and Piper tuberculatum. Phytochemistry. 2000;55:621-6
  • 22
    Navickiene HMD, Bolzani VS, Kato MJ, Pereira AM, Bertoni BW, França SC, Furlan M. Quantitative determination of anti-fungal and insecticide amides in adult plants, plantlets and callus from Piper tuberculatum by reverse-phase high-performance liquid chromatography. Phytochemical Anal. 2003;14:281-4.
  • 23
    Oliveira-Filho EC, Geraldino BR, Coelho DR, De-Carvalho RR, Paumgartten FJ. Comparative toxicity of Euphorbia milii latex and synthetic molluscicides to Biomphalaria glabrata embryos. Chemosphere. 2010;81:218-27.
  • 24
    Orjala J, Erdelmeier CAJ, Wright AD, Rali T, Sticher O. Five new prenylated p-hydroxybenzoic acid-derivatives with antimicrobial and molluscicidal activity from Piper aduncum leaves. Planta Med. 1993;59:546-51.
  • 25
    Rapado LN, Nakano E, Ohlweiler FP, Kato MJ, Yamaguchi LF, Pereira CA, et al. Molluscicidal and ovicidal activities of plant extracts of the Piperaceae on Biomphalaria glabrata (Say, 1818). J Helminthol. 2011;85:66-72.
  • 26
    Schall VT, Vasconcellos MC, Souza CP, Baptista DF. The molluscicidal activity of crown of christ (Euphorbia splendens var. hislopii) latex on snails acting as intermediate hosts of Schistosoma mansoni and Schistosoma haematobium. Am J Trop Med Hyg. 1998;58:7-10.
  • 27
    Silva RV, Navickiene HM, Kato MJ, Bolzani VS, Méda CI, Young MC, et al. Antifungal amides from Piper arboreum and Piper tuberculatum. Phytochemistry. 2002;59:521-7.
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  • 29
    World Health Organization. Schistosomiasis: population requiring preventive chemotherapy and number of people treated in 2010. Wkly Epidemiol Rec. 2012;4:37-44.
  • 30
    World Health Organization. Molluscicide screening and evaluation. Bull World Health Organ. 1965;334:567-81.
  • 31
    World Health Organization. Report of the scientific working group on plant molluscicides. Bull World Health Organ. 1983;12:1-11.
  • 32
    World Health Organization. The role of mollusciciding in schistosomiasis control. Geneva: WHO; 1992. [WHO/SCHISTO/92.107;1992].

Publication Dates

  • Publication in this collection
    Nov-Dec 2013

History

  • Received
    27 Jan 2013
  • Accepted
    2 Apr 2013
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