Schistosomiasis represents a global health concern with over 700 million people at risk of contracting this disease (Weber et al., 2019). In Egypt eradicating this disease is a top governmental priority (Abou-El-Naga, 2018).
Generally, chemotherapy is still one of the most effective methods for controlling
Miltefosine (hexadecylphosphocholine) is an alkyl phospholipid derivative that was developed as a new type of antitumor agent in the 1990s (Eibla and Unger, 1990). Miltefosine has comparative advantage over PZQ as antischistomicidal drug due to its efficacy on the differential developmental stages of
The aims of this work were to compare the efficacy of miltefosine, a chemotherapeutic synthetic compound, with artemether, a natural molluscicidal through light and electronic microscopy and to elucidate their effect on normal immune-histological parameters in
- Group (1): 50 normal control snails (unexposed snails).
- Group (2): 100 normal snails divided into two subgroups (per 50 snails) exposed to sublethal concentrations LC25 of miltefosine and artemether for 2 successive week intervals in two replicates for each drug.
- Group (3):150 snails were exposed to
was further divided into 3 subgroups;
Group (3a): 50 infected snails
Group (3b): 50 infected snails treated with a sublethal concentration LC25 of miltefosine for 2 successive weeks in two replicates for each drug.
Group (3c): 50 infected snails treated with a sublethal concentration LC25 of artemether for 2 successive weeks in two replicates for each drug.
For each drug, the treatment was changed weekly with freshly prepared one to avoid the effect of storage. The snails were exposed to the tested concentrations for 2 successive weeks, then removed from the experimental environment, washed thoroughly with dechlorinated tap water and washed the one time only (after first 24 hours) to recover.
1
2 - The drug artemether was obtained in the form of tablets (Kunming Pharmaceutical Cooperation, PR China) with a documented purity of 99.6%. It is sold under the trade name Riamet and Coartem among others, has a molar mass of 298.374 g/mol and the chemical formula is C16H26O5. The actual concentration was calculated as the percentage of the active material in the used weight. Artemether was applied to snails as aqueous solution of tablets.
A stock solution of 1000 ppm from each drug was prepared on the basis of weight/volume using dechlorinated water. A series of concentrations was prepared for each drug according to the standard procedure recommended by WHO (1965) that allowed us to reach experimental concentrations (LC0, LC10, LC25, LC50 and LC90). The effectiveness of each drug as a molluscicide has been expressed in terms of LC50 and LC90 according to the procedure of Litchfield & Wilcoxon (1949). Three replicates of gradual concentrations from each stock solution were prepared. The snails were exposed to the tested concentrations for 24 hours, then removed from the experimental environment, washed thoroughly with dechlorinated tap water and transferred to aquaria with fresh dechlorinated tap water for the next 24 hours to recover (25 ± 2 oC). Unexposed snails (controls) were assayed side by side with the treated groups under the same laboratory conditions in dechlorinated tap water (WHO,1965). Dead snails were noticed and removed from the container.
Hemolymph samples from snails from all studied groups were collected as previously described by Michelson (1966) by removing a small portion of the shell and inserting a capillary tube into the heart. The hemolymph was collected in a vial tube (1.5 ml) and kept in ice-box The collected hemolymph from infected snails and infected treated groups was used to estimate the total hemocytes count using a Bürker- Turk hemocytometer (while the differential haemocyte count was obtained according to a previously published method (Van der knap et al., 1981).While the differential haemocyte count was obtained on the light microscopy level, hemolymph samples were placed individually onto a clean glass slide. Hemocytes were fixed in 100 % methanol, then stained with Giemsa’s stain examined and counted then photographed using Agfa film according to a previously published method (Abdul-Salam & Michelson, 1980). The hemolymph samples from normal control snails (group1) and drug exposed snails (group 2) were used for transmission electron microscopy examinations (Grimaud et al., 1980).
The results were analysed statistically using the Statistical Package for Social Science (SPSS version 15 package software). Data were expressed as mean (M) ± standard deviation (S. D). The data were statistically analysed statistically significant differences between the treated and the control group using “t” test (Goldstein, 1964).
Bioassay tests results are presented in Table 1, revealing that both compounds has a molluscicidal activity against adult snails. It was noticed that miltefosine showed a marked lethal effect against snails compared to artemether (LC50 and LC90 of 16.88 and 27.97 ppm, respectively). Its LC50 and LC90 values were 13.80 ppm and 24.40 ppm with a slope function value of 1.60. Furthermore, these results demonstrated that the sublethal concentrations (LC0, LC10, & LC25) of miltefosine were lower than the corresponding values of artemether.
Molluscicidal activity of miltefosine and artemether against adult
Tested drug | LC0 | LC10 | LC25 | LC50 | LC90 | Slope |
---|---|---|---|---|---|---|
Ppm | Ppm | ppm | ppm | ppm | ||
Miltefosine | 1.38 | 3.16 | 8.20 | 13.80 | 24.40 | 1.60 |
Artemether | 1.68 | 5.79 | 11.04 | 16.88 | 27.97 | 1.75 |
The recorded data in Figure 1 denoted that the exposure of snails (groups 3b& 3c) to LC25 of miltefosine (8.20 ppm) and artemether (11.04 ppm) for 24 hours for 2 successive weeks exhibited a significant decrease (P < 0.001) in hemocytes count compared with the control group (infected snails; group 3a). The reduction in hemocytes was of 65.48% and 47.62% in miltefosine and artemether treated groups, respectively
As for the number of the different haemocyte types; the exposure of adult infected snails (groups 3b& 3c) to the LC25 drugs concentrations induced a decrease in hyalinocytes percentage. Miltefosine treated–infected snails had the lowest percentage of hyalinocytes (18.40%) compared to artemether treated (26.60%) and control snails (group3a) (52.3%) (Figure 2). On the contrary, both drugs induced an elevation in the percentage of small round cells and granulocytes in corresponding to the infected-control group (group3a).
Hemocytes examination of the normal control snails (group1) by light microscopy revealed the presence of three morphologically different cell types (Plate 1 A-C); small undifferentiated cells with a spherical profile (A), large spherical granulocytes with a double membrane and a relatively large cytoplasm filled with a variable number of basophilic granules (B), and polymorphic hyalinocytes with either a large eccentric nucleus or two nuclei, suggesting atypical cell division (C).
The exposure of adult non-infected
The ultrastructural examination of normal control hemocytes (group1) showed the presence of three morphologically different cell types (Plate 4 A-C). The exposure of adult snails to sublethal concentrations (LC25) of the examined drugs showed a different type of cells with two types of globules in the cytoplasm that appeared only in the hemolymph of treated snails (group 2) (Plate 5 A-C).
Miltefosine treatment induced the following alterations in hemocytes types of non-infected snails (group 2 a): small undifferentiated cells showed a large nucleus, an intact cell membrane, cytoplasmic extensions (pseudopodia), vacuoles, and phagolysosomes in the cytoplasm (5 A); granulocytes presented nucleus with irregular boundaries and an irregular chromatin distribution, the cytoplasm contained phagolysosomes, granules, and some cell organelles such as the mitochondria and rough endoplasmic reticulum (5 B); hyalinocytes presented a degenerated outer cell membrane, the nucleus had an irregular membrane, and the nucleus shrunk in size (5 C).
Artemether induced the following on different types of hemocytes of snails (group 2 b) (Plate 6 A-C): small undifferentiated cells had an intact cellular membrane with extended pseudopodia, the cytoplasm and the nuclei contained phagolysosomes and vacuoles (6 A); granulocytes showed pseudopodia, cytoplasmic granules, and vacuoles in cytoplasm (6 B); while hyalinocytes showed an intact cell membrane, degenerated nuclei, and difficult to identify organelles (6 C).
Internal defense mechanisms of invertebrates depend upon an innate immune system including cellular and humoral components (Le Clec’h et al., 2016). Cell-mediated immune response resulted from the presence of various cell categories that are vital in defense and constitute the primary barrier against invading parasites and bacteria as well as accumulate different substances such as molluscicides, heavy metals and pesticides. These are mobile amoeboid cells referred to as amoebocytes or hemocytes (Bernard, 2016). Furthermore, hemocytes of the snail are used to determine the prepatency period of infection with schistosomiasis (Kamel
In the present study, the hemocytes of
In regard to the types of hemocytes, the present study detected 3 types of cells in
The ultra-structural observation of hemocytes showed that the tested drugs induced morphological alterations, such as irregular nuclear boundaries, irregular chromatin distribution, degenerated nuclei, and vacuolated cytoplasm with electron-dense phagolysosomes; this is in agreement with the results obtained by others (El Sayed et al., 2011; Ibrahim et al., 2018). Kamel
Despite the promising molluscicidal activity of miltefosine, studies show administration results in severe side effects, miltefosine had broad biocide activity as well as it has been shown that miltefosine is a teratogenic agent and its use in the treatment as an anti-Leishmania medication has been associated with severe side effects (Bhattacharya et al., 2007; Eissa et al., 2011). On the contrary, the present study suggests that artemether can be effectively used as a safe plant origin molluscicide in the national
Miltefosine and artemether have a toxic effect on