Efficacy of Miltefosine and Artemether on infected Biomphalaria alexandrina snails with Schistosoma mansoni: immunological and histological studies

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 Schistosoma infection (Bertão et al., 2012a). Since the mid-1970`s, PZQ (praziquantel derivatives) represented the anti-helminthic drug of choice. Despite its efficiency in reducing morbidity and mortality rates in Schistosoma infections (Caffrey, 2007), it is not active against early stages of schistosomes and causes species resistance (Bertão et al., 2012b). Currently, miltefosine and artemether (antimalarial drugs) have a potential application in schistosomiasis treatment (El Beshbishi et al., 2018).

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 S. mansoni in infected mice (Eissa et al., 2011). Miltefosine used as an schistosomicidal drug increased helminths mortality rate and induced extensive tegumental changes in the adult worms of Egyptian and Brazilian strains of S. mansoni in vitro and in vivo (Eissa et al., 2015; El-Faham et al., 2017). In addition, miltefosine presents a molluscicidal activity against both Biomphalaria alexandrina and Bulinus truncatus snails (Eissa et al., 2011). Artemether is a methyl ether derivative of artemisinin, a compound extracted from the leaves of the Chinese wormwood plant (Artemisia annua) (Mossalem et al., 2013). It was first described as anti-schistosomal agent in 1980s, against juvenile worms (5-21day-old) S. japonicum (Liu et al., 2012). Artemether efficiency as a schistosomicidal and molluscicidal agent has been previously shown (Al-Kazzaz et al., 2014; Madbouly et al., 2015).

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 S. mansoni-infected B. alexandrina snails.

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.

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 et al. 2006). Previous experimental studies proved that the parasitic infection induced positive effect upon hemocytes count by increasing their number in the hemolymph of various species of snails (Barcante et al., 2012; Mossalem & Ibrahim, 2019; Suwannatraia et al., 2019).

In the present study, the hemocytes of S. mansoni infected snails (had higher significant increase compared to the treated -infected snail groups with the sublethal concentration (LC₂₅) of miltefosine and artemether as in previous studies (Mossalem et al., 2013; Ibrahim et al., 2018; Mossalem & Ibrahim, 2019). The decrease in hemocytes count in treated snail groups might be a result from tissue damage in digestive and hermaphrodite glands as they participated in tissue repair (Esmaeil, 2009). Another explanation might be the reduction in transaminase enzyme activities, sensitive tools in physiological alterations detection (Kamel et al., 2007), or perhaps, it is the result of hemocyte cells from the hemolymph migrating to connective tissues (Cochennec-laureau, 2003).

In regard to the types of hemocytes, the present study detected 3 types of cells in B. alexandrina hemolymph; hyalinocytes, small round cells and granulocytes. This finding agrees with the results of other studies (Mohamed et al., 2006; Cavalcanti et al., 2012). Some authors suggested that the hyalinocytes and the granulocytes are different cell types, while others considered that they represent different developmental phases of the same cell type (Oliveira et al., 2010). Another study revealed that granulocytes were phagocytic cells with pseudopods capable of encapsulating large particles, while the hyalinocytes were spherical smaller cells without pseudopods (Yoshino et al., 2008). Additionally, the present study showed that the percentage of hyalinocytes decreased in both miltefosine and artemether exposed groups, while the percentage of round small cells (undifferentiated cells) and granulocytes increased in exposed groups compared to the normal uninfected control group. The present data are in accordance with Bakry et al. (2012) with a study showing that methanol extracts of Azadirachta indica plant induced a significant increase in the number of granulocytes in the hemolymph of B. alexandrina snails indicating a high response of the snails against the treatment. Several studies explained the fluctuations in percentage of the types of hemocytes of aquatic snails as a result of drug treatment (Bakry et al., 2012). A study reported that granular hemocytes were the major responsive hemocytes type in B. alexandrina snails treated with a plant growth regulator (Mepiquat chloride) (Mohamed & Abdel-Gawad, 2005). Another study detected an increase in the percentage of small round undifferentiated cells, which may be due to the stimulation of the hematopoietic organ producing undifferentiated hemocytes that differentiated into granulocytes to compensate their reduction in number (El Sayed, 2006). Furthermore, a study stated that hyalinocytes are thought to be responsible primarily for wound repair requiring aggregation at injury site, thus their number decreased in the hemolymph (Barcante et al., 2012). It was reported that granulocyte cells are immunological active cells found mainly in the hemolymph of snails, instead of remaining in the damaged tissue to face external stimuli (Oliveira et al., 2010)

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 et al. (2006, 2007) revealed the presence of a remarkable activation in hemocytes due to a sublethal concentration of plant treatment and molluscicidal compound. They attributed the degenerative changes of hemocytes cellular organelles to a direct toxic effect of these molluscicidal drugs. Further, the continuous exposure to sub-lethal concentrations of artemether caused an increase in glycogen content in hemocytes causing them to increase in size by increasing the time of exposure as reported by Mossalem et al. (2013).

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 S. mansoni control program due to some studies, do not show evidence of harm on non-target organisms. So, further research studies are warranted to evaluate the impact of artemether as safe molluscicidal and schistosomicidal agent (Piola et al., 2010; Elmorshedy et al et al., 2016).

Miltefosine and artemether have a toxic effect on B. alexandrina as they negatively affect immunological processes inducing degenerative changes and fragmentation of hemocytes. Consequently, tested drugs could be ranked as beneficial molluscicidal agents for the control program of schistosomiasis. However, artemether is cheaper to produce and safer for vector control. To further asses artemether in the control of schistosomiasis, it should be evaluated and tested for complete efficacy within the operational research bases for Schistosoma infection control.