Mem Inst Oswaldo Cruz, Rio de Janeiro, 93 (Suppl.I) October 1998
Effects of Euphorbia milii Latex on Schistosoma mansoni Eggs, Miracidia and Cercariae
Laboratório de Toxicologia Ambiental, Escola Nacional de Saúde Pública, Fiocruz
*Laboratório de Biologia e Controle da Esquistossomose, Departamento de Medicina Tropical, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil
The latex of "Crown-of-Thorns" (Euphorbia milii var hislopii) is a potent plant molluscicide and a promising alternative to niclosamide (NCL), today`s mostly used molluscicidal compound (MC Vasconcellos & VT Schall 1986 Mem Inst Oswaldo Cruz 81: 475-476). In addition to being an effective molluscicide, NCL also kills miracidia and cercariae, the two free living stages of Schistosoma trematodes. It is active against other helminths as well. Owing to its prominent cestocide activity NCL has been used in human and veterinary medicine as a drug of choice to treat several tapeworm infections (P Andrews et al. 1983 Pharmac Ther 19: 245-295). However, the effects of E. milii on helminths have not been studied so far. In this study we investigated the effects of E. milii latex on S. mansoni eggs, miracidia and cercariae. Data on the toxicity of NCL to these developmental stages of S. mansoni were also obtained for comparative purposes.
E. milii latex was obtained from plants cultivated in the district of "Ilha do Governador", Rio de Janeiro, Brazil, in July-August, 1995. Lyophilized latex and NCL ethanolamine salt (BayluscideWP70â) were dissolved in dechlorinated water. S. mansoni eggs, miracidia and cercariae were from the "Belo Horizonte" (BH) strain maintained in Biomphalaria glabrata snails and Swiss-Webster mice (WL Paraense & L Corrêa 1989 Mem Inst Oswaldo Cruz 84: 281-288).
Eggs hatching inhibition test - Schistosoma eggs were recovered from infected Swiss mice. Livers were homogenized in NaCl 1.7% w/v. The homogenate was filtered and left to stand for 1 hr. The sediment containing eggs was resuspended in NaCl 1.7% w/v and again left to stand for 1 hr. The procedure was carried out in the dark at 4oC. The eggs were then incubated in the dark at 26oC for 2 hr in the presence of latex (10, 25, 50 and 100 mg/l), NCL (0.05, 0.1 and 10mg/l) or dechlorinated water. Solutions were placed under a tungsten lamp (60 W) during 1 hr for egg hatching. Hatching was stopped by adding an alcohol-formalin-acetic acid solution. The proportions of hatched and unhatched eggs were scored in samples of 30 eggs.
Lethality to miracidia -The sediment containing eggs was resuspended in dechlorinated water and exposed to artificial light (60W). Owing to their phototaxic behaviour, swimming miracidia flocked to the illuminated side-arm of a 150 ml glass flask where they were collected. thirty miracidia per concentration of latex or NCL were then exposed to testing solutions in multi-well plates. The number of living miracidia was recorded after 2, 3 and 4 hr of exposure to testing solutions. All miracidia were tested within 2 hr after hatching.
Lethality to cercariae -Freshly shed cercariae were exposed to testing solutions (200 ± 2 cercariae per concentration) in tissue culture plates (60 x 15 mm) for 4 hr. Evans Blue dye was used to stain dead cercariae. The numbers of living and dead cercariae were recorded. The experiment was repeated three times.
Effect on cercariae infectivity - Freshly shed cercariae were exposed to testing solutions for 2 hr in 5 ml assay tubes. Female Swiss Webster mice (4 to 6 weeks-old) had their tails immersed in assay tubes containing 100 pretreated cercariae for 30 min. Ten mice per concentration of latex or NCL were used. The number of cercariae that failed to penetrate the mouse skin was counted, and all infected mice were killed eight weeks later. Adult worms were recovered from mesenteric veins after a portal-hepatic perfusion.
The present study findings suggested that NCL (0.05 mg/l) does not interfere with S. mansoni eggs hatching. Approximately 50% of eggs hatched during the 3 hr test period in the control group as well as in NCL - and latex-treated groups. No difference between control and treated groups was found. Contrasting with the lack of action on eggs, NCL (0.05 mg/l) proved to be lethal to swimming miracidia (Table I) and cercariae (Table II). No previous investigation of NCL effects on eggs was found in the literature, but its miracidicidal and cercaricidal properties have been observed by several authors. For instance, it was reported that S. mansoni miracidia were killed by NCL at concentrations as low as 0.3 mg/l (within minutes) and 0.1 mg/l (after longer exposures) (Andrews et al. loc. cit.). More recently, a 4 hr-LC50 for NCL miracidicidal effect as low as 0.03 mg/l was obtained by PB Tchounwou et al. (1991 J Environ Sci Health B26: 69-82). On the other hand, it was reported that S. mansoni cercariae were rapidly killed by NCL at concentrations as low as 0.1-0.2 mg/l (Andrews et al. loc. cit). PB Tchounwou et al. (1992 Environ Toxicol Water Qual 7: 107-117) also investigated the cercaricidal effect of NCL and found a 4 hr-LC50 equal to 0.04 mg/l. In the present study, miracidicidal and cercaricidal effects of NCL were noted at a concentration as low as 0.05 mg/l. It is of note that this concentration of NCL is well below its 24hr-LC50 (0.16 mg/l) and 24hr-LC90 (0.31 mg/l) for adult B. glabrata snails (EC Oliveira-Filho 1995 Estudo Ecotoxicológico do Látex Moluscicida da Coroa de Cristo, Euphorbia milii var hislopii, MSc Thesis, Fundação Oswaldo Cruz, Rio de Janeiro, 157 pp.). The data presented in this paper thus suggest that S. mansoni free living stages are killed by NCL at concentrations which are not lethal to their intermediate host snails. However, results also showed that, at such a low concentration of NCL (0.05 mg/l), a relatively long exposure period is necessary to induce cercarial deaths. While exposure of cercariae to NCL (0.05 mg/l) for 4 hr resulted in 68.8% mortality rate in one experiment (Table II), exposure for just 2 hr was ineffective in increasing cercarial mortality in the infectivity test (Figure). Neither the cercarial survival, nor the proportion of cercariae that penetrated the mouse skin was affected after a 2 hr exposure to NCL (0.05 mg/l). On the other hand, none of these penetrating NCL-treated cercariae developed into adult worm in infected mice. These findings suggest that NCL does not just kill cercariae, at higher concentrations and longer exposures, but also render most of the surviving ones incapable of developing into adult worms in the final host. Similar results were also obtained by Tchounwou et al. (1992 loc. cit.) with a different sample of S. mansoni. These authors noted that the percentages of worms recovered from NHI mice infected with cercariae pre-exposed to dechlorinated water alone, and NCL 0.02 mg/l and 0.04 mg/l, were 61.6%, 16.3% and 1.5%, respectively. It should be pointed out that infectivity of their S. mansoni sample (untreated controls: 61.6%,) was higher than that observed with the BH strain used in our experiment (untreated controls: 23%). On the other hand, RMF De-Oliveira (1996 Características Parasitológicas e Perfil Isoenzi-mático de Amostras de Schistosoma mansoni Sambon 1907, MSc Thesis, Fundação Oswaldo Cruz, Rio de Janeiro, 112 pp.) obtained a similar infectivity rate (25.5%) with the same BH strain. As demonstrated by RMF De-Oliveira, infectivity in mice of S. mansoni cercariae varies with the origin of the worm sample used.
E. milii latex (10-100 mg/l) did not show any inhibitory effect on S. mansoni eggs hatching. Furthermore, latex proved to be only slightly toxic to miracidia (Table I) and cercariae (Table II). Miracidicidal and cercaricidal effects of latex were tested at concentrations ranging from 10 to 100 mg/l,i.e. concentrations which are 15 to 150 times higher than the 24 hr-LC90 (0.67 mg/l) for adult B. glabrata snails (Oliveira-Filho loc. cit.). Miracidium mortality was found to be somewhat higher in latex solutions than in untreated controls, but no concentration-effect relationship was observed at any exposure time. Moreover, even at the highest concentration tested (100 mg/l), latex was ineffective in achieving a 100% mortality rate during a 4 hr exposure period (Table I). Similar results were obtained with regard to cercarial lethality. In most instances cercarial mortality was slightly higher in latex solutions than in controls, but again no concentration-effect relationship was found (Table II). Even at the highest concentration of latex tested (100 mg/l) mortality rate was as low as 14.4%, a lethal effect well below that obtained with 0.05 mg/l of NCL (68.8%). Contrasting with the absence of consistent lethal effects of latex on swimming cercariae, a concentration-dependent effect was observed in the cercariae infectivity test (Figure). Neither cercarial survival, nor the percentage of cercariae that succeeded in penetrating the mice skin was reduced by latex (10-100 mg/l, for 2 hr). Nonetheless, the percentage of recovered worms was substantially reduced in mice infected with cercariae exposed to 50 and 100 mg/l of latex. This result suggested that E. milii latex - at least at the two highest concentrations tested - was toxic to S. mansoni cercariae rendering them incapable of developing into adult worms in the final host. From these observations one has to conclude that latex is slightly toxic to S.mansoni free living stages at concentrations much higher than those which are sufficient to kill their intermediate host snails. Thus, in contrast to NCL, E. milii molluscicidal latex does not present the additional advantage of also affecting the viability of schistosoma miracidia and cercariae.
In conclusion, E. milii latex had no effect on S. mansoni eggs and was slightly toxic to miracidia and cercariae. NCL, on the other hand, was effective in killing miracidia and cercariae at a concentration which is not lethal to their intermediate host snails. These findings seems to support the view that, in comparison with the reference molluscicide NCL, E. milii latex has a narrower spectrum of biocidal actions.
Aknowledgments: to the staff of the Department of Malacology, Oswaldo Cruz Institute, Fiocruz, for providing the parasite strain.