Leishmaniasis continues to colonize new regions in many parts of the words due partly to climate changes which allows wide dispersal of both its sand fly vectors and animal hosts. The most prevalent clinical form of the disease, the cutaneous leishmaniasis is increasingly becoming irresponsive to pentavalent antimonial drugs incurring costly, painful and long-lasting cure which usually leaves ugly scars [35]. Therefore, the search for new remedies for the disease particularly among natural products has gained momentum across the world. Many studies have examined the cytotoxic effects of larval excretion/secretion of various flies including L. sericata against different Leishmania species both under in vitro and in vivo conditions [25, 35, 36]. The present study evaluated the anti-leishmanial activity of the crude and fractionated ES of L. sericata against promastigotes and amastigotes of L. major both under in vitro and in vivo conditions using Balb/c mice as an animal model. This study also examined IC50 of the ES products against L. major promastigotes and cytotoxicity of J774A.1 cells to larval products. To the best of the authors’ knowledge, this is the first comparative study dealing with the effects of L. sericata crude ES and its fractions on L. major and macrophage cells.
In this study, the highest rate of cytotoxic effects of ES at the highest concentration used were 15%, 13% and 12% for ES> 10 kDa, ES <10 kDa and crude ES, respectively (Figure 4). This result contrasted the study by Sanei-Dehkordi et al. in which the cytotoxicity of L. sericata larval ES to the same macrophage cell line was reported to be 40%. Although, the ambiguity over the exact concentration of applied ES in their study makes the comparison inappropriate [25]. However, testing L. sericata hemolymph and saliva on the same cell line, [34] reported reduced toxicity to macrophages as in our study[34]. The evaluation of L. sericata and Sarconesiopsis magellanica ES effects on human lung cell line, MRC5, showed that the ES products of the flies had no effects on the survival rates at 10 µg/ml concentration, but they reduced the survival at 20 µg/ml [37]. The toxicity of ES seems to be a function of insect species, rearing methods, ES concentrations and storage conditions, as well as used cell lines.
Larval ES products of L. sericata were effective against promastigotes. The crude ES was more lethal than the fractions; ES> 10 kDa and ES <10 kDa. These findings are consistent with other studies in which the effects of ES, hemolymph, and saliva of L. sericata larvae were evaluated against L. tropica both under in vivo and intro conditions [24, 34]. Similar results have been reported by other authors examining promastigote susceptibility to larval ES products [38, 39].
The antibiotic properties of L. sericata-derived ES were already shown against fungi as well as gram-positive and gram-negative bacteria [40, 41]. In Fact, the ES fractions with molecular weights of <1kDa and 3–10kDa of L. sericata have been shown to exert antibacterial activity against gram-positive and gram-negative bacteria including Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus [42]. The results of this study showed that the ES fraction with molecular weight less than 10 kDa, has lower level of anti-leishmanial activity compared with the ES fraction of higher molecular weight (>10 kDa). However, the crude ES showed the highest toxicity to L. major both under in vitro and in vivo conditions. Therefore, for an effective and strong anti-leishmanial activity, apparently all ES constituents with different molecular weights are necessary.
Susceptibility analysis of intracellular amastigotes of L. major to ES of L. sericata showed that they are more vulnerable to highly concentrated ES than low concentrations. The ES significantly reduced the parasite survival. This finding contrasted those reported by [25, 38] using L. major and L. panamensis amastigotes to infect the macrophage cell line J774 and the U937 cell line respectively. The authors postulated that the applied ES products were more toxic at low concentration that at high concentration. In the present study, the lowest viability percentages of amastigotes were 20.6 ± 2.7 and 15.5 ± 1.1 which induced by treatments with the crude ES (300 µg/ml) and glucantime (100 µg/ml) respectively (Table 1, Figure 5). The survival index values upon treatment with the crude ES were less than those obtained with ES >10 kDa and ES <10 kDa in amastigote-infected macrophage (J774A.1 cells). Also, a considerable reduction in survival index was seen in the treated cells compared to the control cells (Table 1). It is to note that anti-leishmanial effects of the crude ES and its fractions may be maintained by adjusting their concentrations [34, 43]. In the present study, parasite load and survival index were determined in vitro and in vivo. In both cases, the lowest parasitic load was induced by the crude ES as well as glucantime. Also, a significant decrease in the parasite load and survival index were observed in groups treated with larval ES products compared to the negative control (Table 1, Figure 7).
In this study, the crude ES and glucantime performed better in terms of wound size reduction in Balb/c mice infected with L. major averaging at 5 mm2 and 4.6 mm2 respectively (Table 2). There was a statistically significant difference in terms of wound size reduction between mice treated with the crude ES, ES> 10 kDa and ES <10 kDa with those in the negative control. However, no significant difference was observed between Eucerin treated and untreated mice (Figure 6). Using L. sericata maggots directly to treat the lesions of Balb/c mice infected with L. major, [36] failed to record any significant difference between the treated and untreated lesions. This proves that the extracted ES of L. sericata larval was more effective than the debridement activity of the larval in healing the leishmanial wounds. The study by Sanei-Dehkordi et al. [25] has confirmed that the ES extracts of L. sericata and C. vicina larval were highly effective in reducing the lesion size of Balb/c mice infected with L. major when compared with the negative control. A similar result was also confirmed the effectiveness of larval ES of L. sericata in healing the leishmanial ulcers of Balb / c mice infected with L. tropica compared to control group (p < 0.001) [24]. However, another study showed that both maggot therapy and ES derived from L. sericata and S. magellanica larvae were similarly effective in treating hamster lesions caused by L. panamensis [28]. The efficacy of L. sericata larval ES in reducing the development of the leishmanial lesions was attributed to the substance potency in skewing the monocyte-macrophage differentiation from pre-inflammatory to pro-angiogenic [44].
Various studies have shown the potential therapeutic effects of larval ES of different flies on Leishmania parasites both under in vitro and in vivo conditions using different species including L. amazonensis [45], L. tropica [24], L. major [25, 36], and L. panamensis [28]. We also clearly showed the anti-leishmanial activity of larval ES of L. sericata on the intracellular and extracellular forms of L. major parasite both under in vitro and in vivo conditions. We also provided evidence that the larval ES of L. sericata has both topical and systemic therapeutic effects on leishmanial lesions of the model animal.