Bioactivity of miltefosine against aquatic stages of Schistosoma mansoni, Schistosoma haematobium and their snail hosts, supported by scanning electron microscopy
© Eissa et al; licensee BioMed Central Ltd. 2011
Received: 28 March 2011
Accepted: 11 May 2011
Published: 11 May 2011
Miltefosine, which is the first oral drug licensed for the treatment of leishmaniasis, was recently reported to be a promising lead compound for the synthesis of novel antischistosomal derivatives with potent activity in vivo against different developmental stages of Schistosoma mansoni. In this paper an in vitro study was carried out to investigate whether it has a biocidal activity against the aquatic stages of Schistosoma mansoni and its snail intermediate host, Biomphalaria alexandrina , thus being also a molluscicide. Additionally, to see whether miltefosine can have a broad spectrum antischistosomal activity, a similar in vitro study was carried out on the adult stage of Schistosoma haematobium, the second major human species, its larval stages and snail intermediate host, Bulinus truncutes. This was checked by scanning electron microscopy.
Miltefosine proved to have in vitro ovicidal, schistolarvicidal and lethal activity on adult worms of both Schistosoma species and has considerable molluscicidal activity on their snail hosts. Scanning electron microscopy revealed several morphological changes on the different stages of the parasite and on the soft body of the snail, which further strengthens the current evidence of miltefosine's activity. This is the first report of mollusicidal activity of miltefosine and its in vitro schistosomicidal activity against S.haematobium.
This study highlights miltefosine not only as a potential promising lead compound for the synthesis of novel broad spectrum schistosomicidal derivatives, but also for molluscicidals.
Miltefosine (hexadecylphosphocholine) is one of several alkyllysophospholipid derivatives collectively known as alkylphosphocholines that were originally developed as anticancer agents . The biocidal action of miltefosine against Leishmania species was demonstrated in the mid 1980s [2, 3] and since then, trials for its clinical evaluation have led to the licensing of miltefosine for the oral treatment of leishmaniasis in India, Colombia, and Germany [4–6]. Miltefosine is also active against a variety of protozoa, and more and more data have become available on its activity against other Kinetoplastidae (Trypanosoma cruzi and T. brucei) [7, 8], Trichomonas vaginalis, Entamoeba histolytica and several free living amoebas [11–13]. Apart from its antiprotozoal effect, various bioactivities of miltefosine have been reported; it has a broad spectrum antifungal activity , bactericidal activity against Streptococcus pneumoniae and other pathogenic Streptococci, and it is under investigation as a potential therapy against HIV infection . The mechanism underlying broad range bioactivities and the target (s) is still unrevealed. Most recently, miltefosine was reported to have anthelminthic properties. In a study done in 2011 , miltefosine was found to have schistosomicidal activity and showed comparative advantage over PZQ in being effective against in vivo differential developmental stages of Schistosoma mansoni in the mouse model.
Schistosomiasis is one of the most prevalent diseases in the world, with about 200 million human beings infected in 74 countries. It is estimated that 20 million of them have serious forms of the disease or related disabilities, and that 200,000 people die from the disease every year . Chemotherapeutic measures have been the mainstay in the control of this disease . Since 1970, praziquantel (PZQ) has become the drug of choice against the three major human species of schistosomes, Schistosoma mansoni (Sambon), Schistosoma hematobium (Bilharz), and Schistosoma japonicum (Katsurada) [20, 21]. It is a relatively safe, orally administered drug that leads to reduction of the prevalence of schistosomiasis . Consequently a targeted as well as mass drug administration program presently relies heavily on this drug for the control of schistosome-induced morbidity.
With only one drug of choice for treatment and the possibility of development of parasite resistance [23–27], the present situation is dangerous. Therefore, there is a real need for discovery of a new drug.
Though chemotherapy is one of the most effective methods for the control of schistosomiasis , there is a basic need for more selective and efficient molluscicides for the control of the snail vectors. The control of snails is an important means in the combat against this disease. The presently available synthetic molluscicides tend to be generally biocidal affecting many other animals and/or plants in the snail habitat . Therefore, there is a need to search for other molluscicides with strong but specific activity and less harmful to the environment. As miltefosine, was reported to be a promising lead compound for the synthesis of novel anti-schistosomal derivatives with potent activity against in vivo different developmental stages of S. mansoni, this study was carried out to investigate whether a similar activity can also exist against its aquatic stages and its snail intermediate host, Biomphalaria alexandrina, thus being a molluscicide. In addition, to elucidate whether miltefosine can have a broad spectrum antischistosomal activity, a similar in vitro study was carried out on the same stages, the intermediate host, and the adult worm of Schistosoma haematobium the second major human species. Miltefosine efficacy was evaluated on basis of in vitro bioactivity testing supported by scanning electron microscopy.
Materials and methods
Miltefosine (Milteforan®) 2% veterinary oral solution was kindly supplied by Dr. Paolo Bianciardi, Scientific Advisor,Virbac, Italy.
In vitro schistosomicidal bioassay
The schistosomicidal in vitro bioassay used here followed the main procedure previously described by  and . Thus, the schistosome material Schistosoma mansoni (Sambon) and S. haematobium (Bilharz) was obtained from the Schistosome Biological Supply Centre (SBSC), Theodor Bilharz Research Institute (TBRI), Cairo, Egypt. Mature worms were obtained from hamsters (Mesocricetus auratus), percutaneously infected with 350-400 S. mansoni cercariae per hamster 6-7 weeks earlier, and with S. haematobium cercariae 11-12 weeks earlier. The worms were obtained by perfusion using citrated saline, and the recovered worms were washed from blood in small sieves (20 μm mesh) by phosphate buffer. Worms were washed three times with the culture medium which is used for the assay under a sterilized laminar flow chamber. The culture medium used was RPMI 1640 + l-glutamine + 20% fetal calf serum + antibiotics (300μg streptomycin + 300 IU penicillin + 160 μg gentamycin per ml). The bioassay was carried out using 24 wells tissue culture plates. A stock solution 5 mg/ml of the compound was prepared in DMSO immediately before being used. Three pairs of worms, males and females equally were used for each test well in 2 ml medium and 2 replicats were set up for each species in each case. Exposure of worms to a standard concentration of 10 μg/ml of miltefosine (25 uM) was made for 5 days at 37°C ± 0.5°C in 5% CO2 incubator. A pure medium and a medium containing 0.5% of DMSO (vehicle) were used as negative controls, while praziquantel at 10 μg/ml was used as a reference drug. Worms were examined for their viability using a stereomicroscope, and those not showing motility for one minute were considered dead. The mortality rate of worms was calculated after 5 days exposure. The compound was then retested (secondary screen) using the same technique by successive descending dilutions of the solution. The mortality of worms was determined in each case and the LC50 and LC90 were calculated. The statistical program SPSS version 7.5 was used for the calculation.
Larvicidal (ovicidal, miracidicidal and cercaricidal) activity
Eggs, miracidia and cercariae of S. mansoni and S. haematobium were also obtained from SBSC. The eggs were extracted from the intestines of infected hamsters (Mescoricetus aurautus). Miracidia were obtained from cleaned eggs by hatching them in small amounts of dechlorinated tap water. The cercariae were procured from experimentally infected B. alexandrina and B.truncatus snails at 25°C ± 2°C. The eggs, miracidia and cercariae of both Schistosoma spp. were exposed to the LC50 of miltefosine determined below on snails for 30 min., 25 min and 20 min respectively. Sinking down of the miracidia and cercariae with detachment of the tail in the later case give indication of death of these organisms.
The snail material used were Biomphalaria alexandrina (Ehrenberg) and Bulinus truncatus (Audouin), the vectors of S. mansoni and S. haematobium in Egypt respectively. They were also obtained from the colonies maintained in SBSC. Adult B. alexandrina and B. truncatus snails 4-6 mm in diameter and 2-3 mm shell height respectively were used for testing the molluscicidal activity of miltefosine. The snails were fed on boiled lettuce leaves, blue green algae and fish food.
The efficacy of miltefosine against the adult snails was primarily determined using the standard method of World Health Organization recommendations . Thus one liter of solution with a concentration 20 ppm was prepared and 10 snails were added. The snails were maintained in the solution for 24 h at 25°C + 2°C. After exposure, the snails were thoroughly washed and transferred to fresh water for another 24 h for recovery. Two replicas were carried out and two groups of snails were run in fresh dechlorinated water under the same experimental conditions as the negative control. The currently conventional molluscicide (Niclosamide) was used similarly as positive control. At the end of recovery period, the snails were examined for viability, and the dead snails were counted and recorded to calculate the mortality rate. Miltefosine was then retested by the same method using descending concentrations for LC50 and LC90 determination. The statistical program SPSS package version 7.5 was used for calculation.
Scanning electron microscopy study
Eggs, miracidia, cercariae as well as adult worms of Schistosoma mansoni, and the soft body of its snail host, B. alexandrina, exposed to miltefosine, and non exposed samples that served as controls were fixed in a 10% glutaraldehyde and processed for examination by SEM .
All animal studies presented here have been approved by the local government based on national regulations for animal experimentation.
Results & discussion
In vitro schistosomicidal activity
In vitro schistosomicidal activity of miltefosine on Schistosoma mansoni and Schistosoma haematobium adult worms (after 5 days exposure).
Ovicidal, miracidicidal and cercaricidal activities
Ovicidal and schistolarvicidal activity of miltefosine on Schistosoma mansoni and Schistosoma haematobium
Concentration of Miltefosine
Solution (μg/m l)
100% mortality after (minutes)
100% mortality after (minutes)
100% mortality after (minutes)
An SEM study of miracidia has been previously carried out by  and . SEM of S.mansoni miracidium exposed to miltefosine showed loss of cilia from its surface, and the protruded apical papilla showed swollen oedematous corrugated areas. As a result the miracidium loses its ability to swim in water searching for its snail host. It may also lose its capability to penetrate the snails, because of the changes in the apical papilla .
Molluscicidal activity of miltefosine on Biomphalaria alexandrina and Bulinus truncatus snails after 24 hours at 25°C + 2°C.
A review of the literature showed that a number of studies have been carried out on the effect of several compounds and plant extracts on miracidia and cercariae as well as medically important snails with various degrees of success [51–54].
Although, the results of this study demonstrated the molluscicidal activity of miltefosine, its broad biocide activity may makes it unsuitable for snail control. Therefore, this study draws attention to miltefosine as a promising lead compound for the synthesis of more potent and selective molluscicidal derivatives.
This study showed that miltefosine has a schistolarvicidal activity on the different aquatic stages of S.mansoni and S.haematobium, and a lethal in vitro effect on adult worm of both species. In addition, considerable molluscicidal activity was also demonstrated against their snail hosts. These biocidal activities were supported by SEM studies which further strengthen current evidence of miltefosine's activity. To the best of our knowledge, this is the first report of mollusicidal activity of miltefosine and its in vitro schistosomicidal activity against S.haematobium. Thus, this study highlights miltefosine not only as a potential promising lead compound for the synthesis of novel broad spectrum schistosomicidal derivatives but also for molluscicidals.
We are grateful to Dr. Paolo Bianciardi, Scientific Advisor, Virbac, Italy, for providing miltefosine used in this study.
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