The therapeutic effects of larval excretion/secretion of Lucilia sericata on Leishmania major under in vitro and in vivo conditions


 Background

Leishmaniasis is a neglected infectious disease caused by a kinetoplastid protozoan. The disease generally manifests as characteristic skin lesions. Due to the lack of definitive treatment and drugs without side effects, many studies have focused on natural compounds as promising drugs for its treatment. This study aimed to evaluate the effects of larval excretion/secretion products (ES) of Lucilia sericata in crude and fractionated forms on Leishmania parasites under both in vitro and in vivo conditions.
Methods

 In vitro experiments involved evaluation of ES products on both promastigotes and amastigotes inside infected macrophages, whereas in vivo experiments included comparative treatments of Leishmanial lesions of mice using Eucerin-formulated ES products and glucantime.
Results

The IC50 values were 38.7 µg/ml, 47.6 µg/ml, 63.3 µg/ml, and 29.1 µg/ml for crude ES, over 10 kDa ES-fraction, under 10 kDa ES-fraction, and glucantime respectively. Significant differences were observed between viability percentages of promastigotes treated with crude ES and its fractions compared to negative control (p < 0.0001). Crude ES was more effective on amastigote than other two ES fractions at 300 µg/ml concentration. Macroscopic measurement of lesion sizes revealed that the reduction of lesion size in mice treated with crude ES followed quicker cascades of healing than in those treated with glucantime and fractionated ES.
Conclusion

The present study showed that larval ES of Lucilia sericata in both crude and fractionated forms are effective on both intracellular and extracellular forms of L. major. It also provided evidence that the larval ES exerts both topical and systemic therapeutic effects on leishmanial lesions of the model animal.


Introduction
Leishmaniasis is a neglected tropical disease caused by protozoan Leishmania spp. It is estimated that approximately 350 billion people who live in endemic areas are at risk of various forms of leishmaniasis [1]. About 20 species of Leishmania are known to be capable of infecting humans and a range of animals, causing one of three clinical forms in humans called visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), and mucosal cutaneous leishmaniasis (MCL) [2]. Infected bites of Phlebotomus and Lutzomyia female sand ies are the main route of transmission of Old World and New World leishmaniasis respectively [3].
CL is the most common form of the disease which causes dis guring skin lesions with life-long scars on the hands and face [4]. Globally, leishmaniasis imposes a great deal of disability-adjusted life years (DALYs) and economic loss each year [5]. In endemic areas, abundance of sand y vectors, expensive drugs, long treatment time and parasite drug resistance are among serious setbacks for the control of the disease [6,7]. In addition to the side effects of medications, the post-treatment ugly scars of CL leave unaesthetic stigma especially in children especially in girls [8]. So far, glucantime, pentamidine and amphotericin B remains the rst line treatments of CL. To be effective, the drugs need frequent and painful injections. However, upon prolonged use, the drugs may associate with clinical complications such as cardiac arrhythmia and anemia, and even toxicity and renal failure [9,10,11]. Given these problematics, alternative therapies have recently been suggested for CL treatment. These include the use of insect-derived natural compounds such as maggot derived products which showed wound healing effects [12,13].
Maggot debridement therapy (MDT) has been widely used in treating chronic wounds. MDT has been successfully used to treat necrotizing fasciitis, perianal gangrene, surgery wounds, burns, venous, arterial and diabetic foot ulcers [14,15]. The maggots of Lucilia sericata (Diptera: Calliphoridae) are usually used for MDT [16,17]. These maggots exert a combination of wound debridement and disinfection as well as accelerated wound healing by secreting various enzymes for instance proteases and nucleases, antimicrobial peptides (AMPs) and small active molecules [13,18,19]. Since its emergence 20 years ago, MDT is increasingly recognized as a promising alternative therapy for wound healing which results not only in e cient wound debridement but also in risk reduction of post-surgery infections [14,20,21].
Nowadays, MDT has received approval as a medical device in many countries including USA (FDA, 2004, case number K033391) [22].
The larval excretion/secretion product (ES) of Lucilia sericata has exhibited antimicrobial activity against both the gram-positive and gram-negative bacteria [21,23], as well as against protozoan agents of CL [24,25]. In the present study, the effects of crude and fractionated ES of L. sericata were comparatively investigated against Leishmania major parasite both in in vitro and in vivo conditions. Also, in a bid to nd an alternative treatment for CL ulcers, the ES was formulated and tested against leishmanial lesions of infected mice.

Materials And Methods
Collection and rearing of L. Sericata Wild adults of L. sericata were collected using bottle traps baited with raw chicken wings and liver in suburban areas of Saqqez City in Kurdistan Province in Iran during a period between May to July 2020.
The adult ies were anaesthetized with cold shock and morphologically identi ed using a morphological key [26]. The y colony was reared in mesh cages (60×60×60 cm) at the Insectarium of Medical Entomology Department of Tarbiat Modares University under 25 ± 1°C, 60 ± 5% relative humidity and 16: 8 h light/dark cycle conditions [25]. Milk powder and sugar water solution (1: 1 ratio) were supplied to feed the adults. Egg harvesting was performed by placing 150_200 gram of fresh beef liver in the rearing cage for 24 h to allow oviposition [27].

ES collection and sterilization
About 100 stage II and III larval of L. sericata were collected from established colonies. The larval were starved for 6 h before being washed with 0.5% sodium hypochlorite and further with 5% formaldehyde and nally rinsed twice with sterile saline solution in a 50 ml Falcon tube for 5 minutes [28]. Subsequently, 1 ml of saline solution was added to the larvae; the tube was covered with aluminium foil and incubated at 37 ° C for 1 h [28]. The larval ES were then collected by centrifuged at 4000 rpm for 10 minutes [29].
Bradford assay for protein measurement The Bradford Assay Kit was used as a quick and ready-to-use colorimetric method for measuring the total protein of ES. The amount of protein in the solution was measured using a standard curve obtained based on a dilution series of known protein concentrations (0, 31.25, 62.5, 125, 250, 500, 1000 µg/ml). The protein samples were assayed using 7-well plates. The plates were incubated at +25°C for 10 minutes in a dark place. Subsequently, optical absorption was read using an ELISA reader (Model 680, BIORAD Co.) at a wavelength of 570 nm [30].

Fractionation of larval ES
Separation of larval ES fractions into two cutoffs of >10 and <10 kDa was performed using Amicon ultra-4 centrifugal Filer Unit by centrifugation at 7500g for 40 minutes. The isolated fractions and crude ES were passed through 0.22 µm syringe lter for sterilization. The ES products were kept at −20°C until used.
L. major culture conditions L. major strain MRHO/IR/75/ER was maintained by regular passage through Balb/c mice. The amastigotes were isolated from lesions of infected Balb/c mice and transformed to promastigotes on NNN medium. The promastigotes were then cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, USA) and 100 µg/ml penicillin-streptomycin (Thermo Fisher Scienti c, USA). Promastigotes were grown in cell culture asks. The asks were incubated at 26°C until reaching the desired quantity.

Promastigote assay
Promastigotes of L. major were cultured in RPMI culture medium supplemented with 20% FBS in 96-well plates at 1× 10 6 cell/ml concentration. A serial dilution was prepared from crude and fractionated ES at initial concentration of 350 mg / ml using RPMI-1640 medium. The ES dilutions were used to treat promastigotes aliquoted in 96-well plates. Promastigotes cultured in the same medium with no drug added were used as a negative control. Each test was done in triplicate and the plates were incubated at 26°C. In order to evaluate the parasite survival, the multiplication of the promastigotes was determined by counting the cells using a hemocytometer slide (Neubauer chamber) after 24, 48 and 72 h of incubation [31].
Cytotoxicity assay by MTT method

Amastigotes assay
Twelve-well plates were seeded with macrophage cells at 2 × 10 6 cells/ml concentration after a sterile coverslip was placed at the bottom of each well. The plates were incubated at 37°C for 24 h before being infected with L. major promastigotes at 10 parasite/macrophage ratio. The infected plates were then incubated for further 24 h at 37°C under 5% CO 2 atmosphere. Free promastigotes were washed out of wells with PBS and a series of concentrations of 1) crude ES (150-300 µg/ml), 2) ES >10 kDa (150-300 µg/ml), 3) ES <10 kDa (150-300 µg/ml), and 4) glucantime (50-100 µg/ml) were added to the plates based on IC 50 values obtained in the earlier promastigote assays. The 5th group of plates received no treatment as a negative control. After 72h incubation, the coverslips inside the wells were xed with methanol, stained with 10% Giemsa and examined by light microscopy. The number of infected macrophages and the average number of parasites per macrophage were counted per 100 cells [31].

Animal ulcers treatment
Thirty female Balb/c mice (4-6 week old) were obtained from Razi Vaccine and Serum Research Institute (Karaj-Iran). The mice were divided in six groups, each consisting of ve animals. Each group was kept in a separate cage, fed ad libitum in the stress-free Animal House of Tarbiat Modares University.
Logarithmic phase L. major promastigotes were used to prepare an inoculum containing 2×10 6 cells at a nal volume of 100 µl/ml for subcutaneous injection of each mouse at the base of its tail. Injections were performed by insulin syringes under an aseptic laminar air ow cabinet. Lesions were development after 5 weeks' post-inoculation when lesion treatment started by topical application of ES products. The lesion size was measured before and after treatment and used as an indication for wound healing. The weekly measurement of the lesion sizes was continued for further four weeks.

ES preparation for ulcers treatment
The crude ES and its fractions (300 µg/ml) were combined separately with Eucerin at a ratio of (1: 1) to obtain formulated ointments for lesion treatment. The rst group of infected mice was treated with an ointment formulated with the crude ES, the second group with ES fraction >10 kDa and third group with ES fraction <10 kDa. The fourth group was treated with glucantime as positive control and the fth group kept without treatment as negative control. Finally, the sixth group was treated with pure Eucerin to evaluate its possible impact on lesion treatment. All medications were prepared on the daily basis before application.

Parasite load evaluation
Eight weeks after lesion treatment, two mice of each treated group were dissected to estimate the parasitic load in their spleens. To this end, a piece of each spleen was mixed with 500 µl RPMI medium. Then, 50 µl/ml of the mixture was transferred to a 96-well plate containing complete medium enriched with 20% FBS. The plates were incubated at 26°C for 10 days. Each test was performed in triplicate.
Fifteen serial dilutions were prepared for all ve mice groups. Then, the total number of positive wells (presence of motile promastigotes) and negative wells (absence of motile promastigotes) was identi ed by an inverted light microscope [33].

Statistical analyses
Experimental results were analyzed with T-test, and one-way ANOVA using GraphPad Prism version 6.07.
To determine the independence of two categorical variables Chi-square and/or Fisher's exact tests were also undertaken. The data was presented as means ± standard deviation (SD). Dose-response curves were drawn using non-linear regression. Bradford equation was obtained through online software at (https://www.aatbio.com) and the graph was plotted using Prism. Amastigote susceptibility to ES was analyzed using the equations presented in [34]. The difference was considered signi cant at P <0.05.

Bradford assay
As depicted in Figure 1, there was a linear relationship between protein concentrations and the read OD's which were used to calculate the linear equation and therefore the protein concentrations of ES samples.  Amastigote susceptibility to larval ES fractions of L. sericata Table 1 indicates that infection rates of macrophage cells and percentage of viable amastigotes considerably reduced upon treatment with larval ES fractions for 72 h compared to the control group (p = 0.0012 and p = 0.0004, respectively). The reduction in macrophage infection rates induced by ES >10 kDa and ES <10 kDa was similar (p = 0.4763) but signi cantly lower than that caused by glucantime (p = 0.015 and p = 0.007, respectively). However, crude ES and glucantime were similarly effective in reducing macrophage infection rates so that the difference between them was not statistically signi cant (p = 0.7486). In addition, no difference was observed between the mean number of amastigotes per an infected macrophage cell upon treatment with ES >10 kDa and ES <10 kDa (p = 0.290), but the ES fractions were signi cantly less effective in reducing the number of amastigotes in macrophages than glucantime (p = 0.029 and p = 0.010, respectively). In this respect, the crude ES and glucantime exerted similar effects (p = 0.7890). The results showed that the treatment with the crude ES at higher concentrations reduces the infection rate of macrophages and the viability of amastigotes (p = 0.0357).
The treatment with the crude ES at 300 µg/ml concentration lead to a signi cant reduction of the parasite load in macrophages equal to 1.75 ± 0.05 per cell compared with the negative control treatment. The positive treatment with glucantime at 50 µg/ml concentration also reduced the parasite load to a level of 1.96 ± 0.07 amastigote per cell. The survival index of amastigotes recorded the lowest value equal to 53.72 ± 2.44 upon treatment with the crude ES at 300 µg/ml concentration (Table 1 and Figure 5).

Effect of larval ES fractions on leishmanial lesions
Cutaneous lesions in all infected mice began with redness and swelling at the site of inoculation in the third week post-infection. The swelling increased gradually, crust formation occurred and gangrene infection started to develop in the fourth week. The mean lesion size measurements are presented in Table 2. The results of the lesion size measurement showed that the mean lesion sizes in control mice increased progressively to 12.8 ± 2.86 mm 2 until the fourth week post-inoculation. Likewise, the size of wounds increased in Eucerin-treated mice to reach 12 ± 1.87 mm 2 for the same period. In other words, no statistically signi cant difference was observed between the lesion sizes in control and Eucerin groups (p = 0.7824). This shows that Eucerin has no therapeutic effect by its own. In contrast, the lesion sizes started to decrease gradually in groups treated with ES. The mean lesion sizes were 2.6 ± 1.19 mm 2 in the crude ES-treated group, 5 ± 2.35 mm 2 in ES >10 kDa-treated group, 5.2 + 2.280 mm 2 in ES <10 kDatreated group and 2.2 ± 1.327 mm 2 in glucantime-treated group. While the crude ES and glucantime were similarly effective in reducing the lesion sizes with no signi cant difference between them (p = 0.4899), both showed signi cant differences with ES >10 kDa and ES <10 kDa (p < 0.0001) in the same respect. In fact, the latter treatments were similarly less effective against lesion development recording no signi cant difference in reducing lesion sizes (p = 0.7472). After the termination of treatment period, the measurement of lesion size was continued for further weeks to assess the possibility of recurrence. A slight increase of lesion sizes was observed in the case of the crude ES-treated (3.0 ± 2.55 mm 2 ) and glucantime-treated (2.6 ± 1.96 mm 2 ) groups. Whereas the wounds erupted in mice upon the cessation of treatments with ES >10 kDa (6.6 ± 3.78 mm 2 ) and ES <10 kDa (7 ± 3.39 mm 2 ) ( Figure 6).

Parasite loads
Page 10/19 The spleen parasite load of mice infected with L. major was determined using the parasite-limiting dilution assay. The parasite load was signi cantly reduced in treated groups compared with the negative control group (p < 0.0001). The latter group showed no signi cant difference with the Eucerin-treated groups (p >0.05). The crude ES-treated group had the lowest level of parasite load among all treated group with signi cant difference with others (p < 0.0001) except with glucantime treated group (p = 0.2666). As usual, there was no statistically signi cant difference between groups treated with ES >10 kDa and ES <10 kDa (p = 0.0104) (Figure 7).

Mice mortality during study phases
The mortality of experimental mice was monitored from the beginning of treatments until 20 weeks' posttreatments. Mortality in the negative control and Eucerin treated mice started after the seventh week postinfection with sharp rate so that all mice were dead by week 13th post-infection. Whereas, in treated mice, mortality was observed to occur after week 13th post-infection so that more than 60% of mice were alive by the end of week 20th. The mortality in the negative control and Eucerin treated groups were similar with no signi cant difference between them (p = 0.8134). However, both differed signi cantly from other treated group in term of mortality rates (p < 0.0001). The lowest mortality rate was observed among the crude ES treated group, however, its difference with glucantime-treated group was not signi cant (p=0.2396). By the end of 20th week post-infection the survival rates of mice treated with the ES >10 kD and ES <10 kDa were recorded alike at 20% with no signi cant difference among the groups (p = 0.3461), though they differed signi cantly with the crude ES and glucantime treated groups (p < 0.0001) ( Figure   8).

Discussion
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 y 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 ies 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 IC 50 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 rst 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 ies 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 ndings 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 grampositive 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 signi cantly reduced the parasite survival. This nding 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 signi cant 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 mm 2 and 4.6 mm 2 respectively (Table 2). There was a statistically signi cant 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 signi cant 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 signi cant 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 con rmed 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 con rmed 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 e cacy 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 prein ammatory to pro-angiogenic [44].
Various studies have shown the potential therapeutic effects of larval ES of different ies 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.

Conclusion
For the rst time in this work, the larval ES of L. sericata were fractionated into 2 substances of different molecular weights; above 10 kDa and below 10 kDa. This study showed that both fractions are effective in microscopic and macroscopic evaluation of L. major on both intracellular and extracellular forms of the parasite. However, the fraction above 10 kDa had a better effect than the fraction below 10 kDa.
However, the crude ES showed a higher activity compared to the fractionated ES. This study revealed that L. sericata crude ES and its fractions are effective candidates for curing lesions induced by L. major. However, adding suitable adjuvants may reinforce their effects which requires further studies in the future. 45. Arrivillaga J, Rodríguez J, Oviedo M. Evaluación preliminar en un modelo animal de la terapia con larvas de Lucilia sericata para el tratamiento de la leishmaniasis cutánea. Biomédica. 2008;28 2:305-10. Figure 1 Bradford assay standard curve and the plotted ES sample concentrations.

Figure 2
Dose-response curves of tested ES products of L. sericata larvae and glucantime against L. major promastigotes (IC50) at 24 h, 48 h and 72 h intervals.    Progress of leishmanial lesion sizes in treated and control Balb/c mice followed from pre-intervention stages until 8th week post-treatment.

Figure 7
Spleen parasitic loads of mice infected with L. major at 8th week post-treatments. (The data present Means ± SD of triplicates).

Figure 8
Survival records of treated and untreated mice over a period of 20 weeks post-inoculation with L. major promastigotes.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. ESgraphicalabstract.tif