Reviewed by Felio Bello, Ph.D.
Journal Citation
Sanei-Dehkordi A, Khamesipour A, Akbarzadeh K, Akhavan AA, Miramin-Mohammadi A, Mohammadi Y5, Rassi Y, Oshaghi MA, Alebrahim Z, Eskandari SE, Rafinejad J. Anti Leishmania activity of Lucilia sericata and Calliphora vicina maggots in laboratory models. Exp Parasitol. S0014-4894(16)30165-5.
Published Abstract
Use of sterile fly larvae (maggots) of blow flies for the treatment of many different types of skin and soft tissue wounds is called Maggot debridement therapy (MDT). The larvae of blow flies secrete a broad spectrum of compounds with diverse mechanisms of action in the gut and salivary glands called excretion/secretion (ES) products which showed to have antimicrobial activities against Gram negative and positive bacteria. Cutaneous leishmaniasis (CL) which is the common form of leishmaniasis is difficult to treat.
In this study, the effect of ES from 2nd and 3rd stages of L. sericata and C. vicina larvae on in vitro Leishmania major amastigote growth in macrophage was evaluated. The effect of ES on Leishmania growth was estimated by assessing the rate of macrophage infection and the number of amastigotes per infected macrophages. In addition, the anti Leishmania activities of larval and ES of L. sericata and C. vicina on the skin lesion induced by L. major infection was evaluated in susceptible BALB/c mice.
The results showed that ES of both flies reduced the number of infected macrophages; 38 2.6 and 1.5-fold using L. sericata ES and C. vicina ES, respectively, and inhibited amastigotes growth in macrophages; 2.03 and 1.36-fold by L. sericata ES and C. vicina ES, respectively as compared to the control group.
The results showed that L. sericata ES was significantly more effective than C. vicina ES to inhibit in vitro L. major amastigotes growth, The size of lesion was significantly smaller in Balab/c mice treated with L. sericata ES than treated with C. vicina ES. The results of in vivo experiments suggested that pre-treatment with ES derived from L. sericata may have some protective effects on the development of L. major lesion. Therefore, it seems that maggot ES might be considered as a possible candidate for the treatment of cutaneous leishmaniasis.
Context (briefly, why and how the research was carried out)
This work highlights the relevance of leishmaniasis as a worldwide public health problem; the disease is endemic in 98 tropical and subtropical countries, including Iran (the authors are Iranian). The World Health Organization (WHO) has estimated that one tenth of the world’s population is at risk of becoming infected, with a prevalence of 12 million and a yearly incidence of 1-1.5 million for the cutaneous form (this being the most common, which represents more than 99% of cases). The incidence / prevalence of the visceral form is 0.5 million. This disease in its various clinical manifestations (cutaneous, mucocutaneous, diffuse cutaneous and visceral) is considered by the WHO to be one of the 15 neglected tropical diseases. The pathology’s etiological agents are protozoan parasites from the genus Leishmania, transmitted by the bite of a female sand-fly from the genus Lutzomyia in the New World and from the genus Phlebotomus in the Old World. Zoonotic cutaneous leishmaniasis is caused by L. major in Iran while the anthroponotic cutaneous form is induced by L. tropica (LT); however, 90% of cases reported in most Iranian provinces are due to zoonotic cutaneous leishmaniasis.
Pentavalent antimonials currently represent the first-line drug for leishmanial treatment; however, their toxicity, high cost, the prolonged treatment involved, multiple injections and resistance produced in the parasites justify the search for new therapeutic strategies. New drugs have been tried, such as ketoconazole, paromomycin, allopurinol, mefloquine, gentamycin and verapamil, but they have had limited efficacy. Faced with such a negative panorama, larval therapy would seem to be a promising alternative for treating this disease, initially against its cutaneous form (i.e. that with the greatest prevelance).
This work was performed using in vitro and in vivo models for evaluating the effect of ES and larvae of two blowfly species: Lucilia sericata and Calliphora vicina. The first methodological approach involved using a mouse macrophage-derived cell line (J774-A) as substrate for developing infection by the Leishmania major strain, and observing for cytotoxicity when exposed to different concentrations of ES from second and third instars of both blowfly species. Two criteria were used for evaluating the extent of ES action on intracellular amastigotes: the mean number of infected macrophages per 100 counted macrophages, and the mean number of amastigotes per 100 counted macrophages.
Female 6-8 week-old BALB/c mice were used in the in vivo model. The animals were randomly divided into 10 groups (G), each group consisting of 10 mice. The parasite was subcutaneously inoculated into all the groups of mice (2 x 106 promastigotes in 50 µl in the hind left footpad, taken during stationary phase). The development of lesions in groups 1-8 (G1-G8) was treated with ES (G1, G2, G3, G4, G7 and G8) whilst larval therapy (free larvae) was used for treating cutaneous leishmanial lesions in groups 5 and 6 (G5 and G6). G1 and G2 were pre-immunized before inoculating the parasite and G3 and G4 received an inoculation of a mixture of ES and promastigotes before receiving treatment. Group 9 (G9, positive control) received standard treatment with con melamine antimonite (Glucantime) and group 10 (G10, negative control) did not receive any type of treatment. The odd-numbered groups were treated with ES and/or L. sericata larvae and even-numbered groups received C. vicina-derived treatment. The area of the lesions was measured and compared to that of the controls for evaluating treatment effectiveness.
Findings
The authors demonstrated that the optimum non-toxic effect on macrophage cells was obtained with the lowest ES concentration (5%), giving 98% cell viability. Probit analysis gave 16.32% cytotoxic concentration value for L. sericata ES at 50% (CC50) and 40.11% at 90% (CC90) and 18.7% CC55 and 48.01 CC90 for C. vicina acting on the J774-A cell line.
When evaluating the effect of ES derived from both blowflies, L. major mouse macrophage cell (J774-A line) infection assays revealed significant inhibition of the average number of infected macrophages and the average number of amastigotes per macrophage (at 1.25%, 2.5% and 5% concentrations from L. sericata and just 5% from C. vicina) (p<0.05), compared to control group. However, 5% ES concentration (taken from the larvae from both blowfly species) was seen to be the most effective in reducing the amount of infected macrophages. L. major IC50 values regarding amastigote infection rate were 0.84% for L. sericata ES and 1.36% for C. vicina.
Experimental results regarding measuring lesion development in the mice (following inoculation of the parasite in the study groups for 15 consecutive weeks) showed that G4 had the largest average lesion size and G1 the smallest average size. Comparing leishmanial lesion development in BALB/c mice from the study groups with control G10 revealed that average lesion size in groups G1, G3, G5, G6, G7, G8 and G9 was smaller than that for the control group. Except for group G6, there were significant differences between average lesion size for the forgoing groups compared to control group lesion size (p<0.02). Contrarily, the results for groups G2 and G4 gave greater average values regarding size compared to group G10. However, there were no significant differences between groups G4, G5 and G6 compared to control group (p>0.24).
Discussion
The authors demonstrated in this work that L. sericata-derived larval ES had a significant effect on L. major in both in vitro and in vivo conditions. L. sericata was seen to have greater effectiveness compared to the anti-parasitic effect of C. vicina-derived ES. Thereby, for example, the concentration of 5% ES from L. sericata reduced the number of infected macrophages about 2.6, while that for C. vicina was of 1.5 fold. Besides, the inhibition growth of amastigotes in macrophages was 2.03 and 1.36-fold reduction when treated with ES of L. sericata and C. vicina respectively. According to the model in vivo, significant differences were registered among the study groups in the mean lesion size on the mice during 15 weeks (F: 10.68, p: .0001 after Greenhouse-Geisser correction). In general, the results showed that the effect of L. sericata ES on decreasing the size of the lesion was greater than that seen with the use of the free larvae. In vivo results suggested that pre-treatment with ES from L. sericata may have some protective effects on the development of L. major lesions, asthe size of the lesions were smaller in this model.
This is an interesting study as it evaluated (for the first time) larval ES action on the parasite L. major by comparing the effect of the indicated substances and individual larvae from two necrophagous blowfly species. C. vicina was used for the first time for evaluating the anti-parasitic effect of its larvae and ES. A cell substrate (mouse J774A macrophage-derived line) was also used for determining the effect of ES on the parasite’s amastigote forms. The effect of L. sericata free larvae on lesions caused by L. amazonensis had previously been evaluated in Syrian hamsters (Mesocricetus aureatus) (Arrivillaga et al., 2008) and ES and free larvae on L. tropica (LT) promastigotes and amastigotes in in vitro and in vivo conditions (BALB/c) (Polat et al., 2012). Maggot therapy efficacy was also demonstrated in healing the lesions of some human patients which were resistant to meglumine antimoniate treatment, a better response and cure of leishmanial ulcers being obtained with maggot therapy in less time (Polat and Kutlubay, 2014).
The findings and conclusions drawn from this research are consistent with that of previous work (cited above): larvae and larval ES demonstrate anti-Leishmania activity in the models tested and in human patients. There are still some unanswered questions concerning the action of larvae and their ES, especially in terms of their effect on healing cutaneous lesions with or without eliminating the parasite. Future research should address the mechanisms of action by which larvae and/or ES affect Leishmania, and which substances in the ES participate in that anti-parasitic action. Such research should be supported by an integral methodological approach evaluating the macroscopic aspects of larval ES and larvae action on the leishmanial lesions in the selected animal model, the events occurring in the tissue of the lesions being treated (histopathology), and the effect of the substances derived from the larvae and actual larvae on the parasite during treatment. Our group recently carried out work with this approach (Cruz-Saavedra et al 2016.); however, it is necessary to do several methodological adjustments in order to validate the larval therapy in the treatment of lesihmanisis in human patients. For instance, the most appropriate approach would be to quantify parasite load by RT-PCR as it has been shown to be sensitive, specific and reproducible for this type of analysis applied to work with Leishmania parasites.
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