In vitro antischistosomal activity of venom from the Egyptian snake Cerastes cerastes

Introduction: We studied the potential in vitro antischistosomal activity of Cerastes cerastes venom on adult Schistosoma mansoni worms. Methods: Live specimens of the horned viper snake, C. cerastes were collected from the Aswan Governorate (Egypt). Venom was collected from snakes by manual milking. Worms of S. mansoni were obtained from infected hamsters by perfusion and isolated from blood using phosphate buffer. Mortality rates of worms were monitored after 3 days of exposure to snake venom at LC50 and various sublethal concentrations (10, 5, 2.5μg/ml). Scanning electron microscopy was used to investigate tegumental changes in treated worms after exposure to LC50 doses of venom. Results: The LC50 of C. cerastes venom was 21.5μg/ml. The effect of C. cerastes venom on Schistosoma worms varied according to their sex. The mortality rate of male and female worms after 48-h exposure was 83.3% and 50%, respectively. LC50 of C. cerastes venom induced mild to severe tegumental damage in Schistosoma worms in the form of destruction of the oral sucker, shrinkage and erosion of the tegument, and loss of some tubercle spines. Conclusions: The present study demonstrated that C. cerastes venom exerts potential in vitro antischistosomal activity in a time and dose-dependent manner. These results may warrant further investigations to develop novel schistosomicidal agents from C. cerastes snake venom.


INTRODUCTION
Schistosomiasis is a major parasitic disease of the tropics, causing acute and long-term clinical syndromes (1) (2) .Among schistosomicidal drugs, praziquantel is the drug of choice in most areas of endemicity owing to its superior effi cacy (3) .A small number of cases of resistance to treatment with praziquantel have been reported, and represent a major barrier to the increasing global attempts to eliminate the public health burden of schistosomiasis (4) .Such resistance and the possibility of evolving future resistance highlight the need to develop new schistosomicidal treatments (2) (3) (4) .
Previous studies have explored the use of toxins as potential therapeutic agents for treating various conditions such as hypertension (5) (6) , thrombosis (7) (8) , and cancer (9) (10) (11) (12) (13) (14) .Among these studies, the initial interest in snake venom was to evaluate how combat the effects of snakebites in humans and to study how snakebite toxins exert their effects.Crude venoms have also been used in traditional medicine for the treatment of human disease from the early twentieth century (15) .In addition, antiparasitic effects of snake venom have been reported against Leishmania (16) (17) , Trypanosoma cruzi (18) (19) , and Plasmodium (20) (21) parasites.
Cerastes cerastes (family Viperidae), or the horned viper snake, is a medically important and dangerous species of snake in North Africa, and its venom is characterized by its ability to induce severe cytolytic effects (22) .Cerastes cerastes venom is highly toxic, primarily attributable to the activity of various proteolytic enzymes it contains (23) (24) .Cerastes cerastes venom also exhibits a range of biological activities including antiangiogenic (25) , antitumor (26) , antibacterial (27) , and antiprotozoal (16) effects.Therefore, the present study was performed to evaluate the in vitro antischistosomal activity of C. cerastes venom on adult Schistosoma mansoni parasitic worms.To the best of our knowledge, this is the fi rst study of its kind and it may lead to further research on the characterization of novel antischistosomal agents from snake venom.

Snake venom collection and preparation
Live specimens of horned viper snakes, Cerastes cerastes (n = 10), were collected in the Aswan Governorate (Egypt) and identifi ed at the Zoology Department, Faculty of Science, Suez Canal University, Ismailia, Egypt.Without harming the snakes, venom was collected by manual milking and all snakes were then returned to their natural habitat.The collected venom was pooled, lyophilized, and stored at -20°C until use.

Ethical considerations
Hamsters were obtained from the Theodor Bilharz Institute, Giza, Egypt and housed under controlled conditions of humidity and a 12h light/dark cycle with free access to food and water.All experimental procedures involving animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (http://www.nap.edu/catalog/12910.html) and approved by the Research Ethics Committee (Section of Experimental Animals 1/2015) of the Faculty of Science, Suez Canal University, Ismailia, Egypt.

In vitro preparation of worms
Schistosoma worms were obtained from infected hamsters by perfusion and were isolated from blood using small sieves (20µ mesh size) and phosphate buffer.Next, the worms were rapidly washed under sterile laminar fl ow conditions in Roswell Park Memorial Institute (RPMI) culture medium containing 20% fetal calf serum, 300mg streptomycin, 300U penicillin and 160µg gentamicin.Worms were then exposed to a range of sublethal concentrations of snake venom in sterilized tissue culture plates (28) .

Determination of LC 50
A stock solution (500µg/ml) of snake venom was prepared in dimethylsulfoxide (DMSO) and diluted with RPMI 1640 to produce 2ml of working solution with a fi nal concentration of 100µg/ml.Serial concentrations of 10, 20, 30, 40 and 50µg/ml, to cover the range of 0-100% mortality, were used to calculate the lethal concentration 50 (LC 50 ).Mortality rate was calculated as the number of dead worms divided by the total number of worms.LC 50 was estimated using the Statistical Package for the Social Sciences (SPSS) statistical program (version 20).

Viability of worms
Worm viability was evaluated using a stereomicroscope after 24h of treatment or for three successive days of treatment to determine the LC 50 and sublethal concentrations, respectively.Worms showing no signs of motility for one min, in addition to those showing deformities such as blackening, twisting, and contracting, were considered dead.

Mortality rates of Schistosoma mansoni worms
Two replicates, each containing three pairs of worms (equal ratio of males and females) were placed in control and test solutions (21.6, 10, 5 and 2.5µg/ml of venom) at 37°C (29) .

Electron microscopy
Treated worms were fi xed with 4% glutaraldehyde in 0.2M sodium cacodylate buffer (pH 7.3) for 4h, followed by postfi xation in osmium tetraoxide (OSO 4 ) for 2h.Next, the worms were rinsed three times in the same buffer and dehydrated through a series of graded ethanol concentrations from 10-100% for 10 min in each concentration, except for the fi nal concentration (100%) (30 min, 10 min per change).Further dehydration was performed by critical point drying in liquid carbon dioxide.Worms were mounted on copper stubs using double-sided adhesive tape, coated with gold using an S150A sputter coater (Edwards, UK), and viewed with a scanning electron microscope (JXA-840A Electron Probe Microanalyzer; JEOL, Tokyo, Japan).
Table 1 shows the mortality rates of S. mansoni worms after 3 days of exposure to LC 50 (21.6µg/ml)and sublethal concentrations (10, 5, and 2.5µg/ml) of venom.Total mortality rates of worms at LC 50 were 16.7%, 66.7%, and 66.7% on days 1, 2, and 3 post exposure, respectively.Total mortality rates of worms at concentrations of 10, 5, or 2.5µg/ml were 8.3%, 15.5%, and 27.4%; 4.2%, 8.3%, and 20.8%; and 4.2% at days 1, 2, and 3 post exposure, respectively.The susceptibility of male and female worms to the venom differed, with male worms being more susceptible than females were to LC 50 or to sublethal concentrations (2.5, 5, and 10µg/ml).In addition, LC 50 dose had no effect on female worms after 24h but caused 50% mortality after 48h.The sublethal concentrations, 10 and 5µg/ml, had no effect on female worms after 24h but caused 25% and 8.3% mortality among female worms after 72h, respectively.No mortality was recorded among female worms exposed to 2.5µg/ml for 3 days, while the mortality rate among male worms under the same conditions was 8.3%.

Electron microscopy
Surface ultrastructure alterations were observed in adult male and female S. mansoni worms following exposure to LC 50 doses of venom (Figure 2).Mild to severe tegumental damage was noted among both male and female worms.In male worms, marked destruction of the oral sucker, shrinkage and erosion of the tegument, loss of several tubercle spines, and formation of protuberances in the gynaecophoric canal tegument were observed (Figures 2A, 2B and 2C).In female worms, mild to moderate shrinkage and erosion of the dorsal and ventral tegument were observed (Figures 2C, 2D and 2F).

DISCUSSION
To our knowledge, the present study is the fi rst to evaluate the effect of C. cerastes venom against S. mansoni.The LC 50 of C. cerastes venom in S. mansoni worms after 24-h exposure was 21.6µg/ml, a signifi cantly higher dose than that of the reference drug praziquantel (6,675µg/ml) (30) or of combination artemisinin-naphthoquine phosphate treatment (CO-ArNp) after 72h (7.2µg/ml) (31) .In addition, the LC 50 against schistosomes of mefl oquine and hemin alone was determined to be 6.5μg/ml and 232μg/ml, respectively, while the LC 50 of hemin in the presence of mefl oquine (1μg/ml) was 23.2μg/ml (32) .
Disparities in drug susceptibility between male and female S. mansoni worms have been previously reported in several in vitro activity trials (34) .The results of the present study showed that the susceptibility of male and female worms to venom varied, with male worms being more susceptible than females were.In this regard, male S. mansoni worms were also more susceptible than females were to praziquantel, ginger extract, diamines, and amino alcohols and to treatment with certain essential oils (35) (36) (37) (38) (39) .However, there was no differential sensitivity to piplartine between male and female worms in vitro (34) .In contrast, treatment with other compounds such as amino alkanethiosulfuric acids (40) , 2-(butylamino)-1-phenyl-1-ethanethiosulfuric acid (41) , and artesunate (42) produced higher mortality rates among female S. mansoni worms than among males.
Tegumental changes produced by antischistosomal drugs are a necessary mechanism for causing the death of the worm (43) .Generally, if a drug induces tegumental alterations, the onset and precise pattern of these alterations depend on the particular drug and the treatment regimen used (44) .The characteristic features of tegumental damage observed in this study, including destruction of the tegument and erosion and loss of spines, were consistent with those previously described in the literature following in vitro exposure to different antischistosomal agents such as hypericin (45) , allicin (46) , nerolidol (47) , phthalyl thiazole (LpQM-45) (48) , β-lapachone (49) , and DNK (32) .Hence, reductions in worms may be attributed to the tegumental damage observed.
In conclusion, the present study demonstrated that C. cerastes venom exerts potential in vitro antischistosomal activity against S. mansoni in a time-and dose-dependent manner.Further studies are needed, however, to characterize the active compounds present in the venom and their cytotoxic effects for potential use in the future development of novel therapeutic agents.

FIGURE 1 .
FIGURE 1. Mortality rate of male and female Schistosoma mansoni worms exposed to different concentrations of Cerastes cerastes venom for 24h in vitro.

FIGURE 2 .
FIGURE 2. Surface ultrastructure alterations in adult male (A-C) and female (D-C) Schistosoma mansoni worms following incubation with LC 50 (21.6µg/ml) of Cerastes cerastes snake venom.Black arrows indicate destruction of the oral sucker and tegument (A), loss and destruction of tubercle spines (B), formation of protuberances and shortening of spines in the tegumental area around the gynaecophoric canal (C), and mild to moderate destruction, shrinkage, and erosion of the tegument of female worms (D-F).LC 50: lethal concentration 50.

TABLE 1
Mortality rate (%) of Schistosoma mansoni worms exposed to different concentrations of Cerastes cerastes venom for 3 consecutive days in vitro.