Transmission blocking activity of a standardized neem (Azadirachta indica) seed extract on the rodent malaria parasite Plasmodium berghei in its vector Anopheles stephensi

Abstract

Background: The wide use of gametocytocidal artemisinin-based combination therapy (ACT) lead to a reduction of Plasmodium falciparum transmission in several African endemic settings. An increased impact on malaria burden may be achieved through the development of improved transmission-blocking formulations, including molecules complementing the gametocytocidal effects of artemisinin derivatives and/or acting on Plasmodium stages developing in the vector. Azadirachtin, a limonoid (tetranortriterpenoid) abundant in neem (Azadirachta indica, Meliaceae) seeds, is a promising candidate, inhibiting Plasmodium exflagellation in vitro at low concentrations. This work aimed at assessing the transmission-blocking potential of NeemAzal(R), an azadirachtin-enriched extract of neem seeds, using the rodent malaria in vivo model Plasmodium berghei/Anopheles stephensi.

Methods: Anopheles stephensi females were offered a blood-meal on P. berghei infected, gametocytaemic BALB/c mice, treated intraperitoneally with NeemAzal, one hour before feeding. The transmission-blocking activity of the product was evaluated by assessing oocyst prevalence, oocyst density and capacity to infect healthy mice. To characterize the anti-plasmodial effects of NeemAzal(R) on early midgut stages, i.e. zygotes and ookinetes, Giemsa-stained mosquito midgut smears were examined.

Results: NeemAzal completely blocked P. berghei development in the vector, at an azadirachtin dose of 50 mg/kg mouse body weight. The totally 138 examined, treated mosquitoes (three experimental replications) did not reveal any oocyst and none of the healthy mice exposed to their bites developed parasitaemia. The examination of midgut content smears revealed a reduced number of zygotes and post-zygotic forms and the absence of mature ookinetes in treated mosquitoes. Post-zygotic forms showed several morphological alterations, compatible with the hypothesis of an azadirachtin interference with the functionality of the microtubule organizing centres and with the assembly of cytoskeletal microtubules, which are both fundamental processes in Plasmodium gametogenesis and ookinete formation.

Conclusions: This work demonstrated in vivo transmission blocking activity of an azadirachtin-enriched neem seed extract at an azadirachtin dose compatible with 'druggability' requisites. These results and evidence of anti-plasmodial activity of neem products accumulated over the last years encourage to convey neem compounds into the drug discovery & development pipeline and to evaluate their potential for the design of novel or improved transmission-blocking remedies.

Figure 1

Light microscope images (400×) of Plasmodium berghei oocyst development stages (day 10 after mosquito infection). Immature oocyst, with uniform content, A; immature oocysts, with sporoblasts and budding sporozoites, B1 and B2; mature oocyst, with fully developed sporozoites visible, C.

Figure 2

Effects of the neem products on P. berghei midgut stages (zygotes to ookinetes). Parasites were counted on 300 microscopic fields (1000× magnification) of midgut content smears of A. stephensi females that had fed 18 (A), and 20 (B) hours before on mice treated with the indicated neem products and doses. NA25, NeemAzal^® 25 mg/kg; NA50, NeemAzal^® 50 mg/kg; AZA50, azadirachtin 50 mg/kg. Geometric means of zygote + ookinete densities, evaluated over ten smears (30 mosquitoes), with 95% confidence intervals (green bars; left axis). Mature ookinetes/total midgut forms ratio (orange bars; right axis). * means differ significantly from control (Student's t test; p ≤ 0.002)

Figure 3

Light microscope images (1000×) Giemsa stained P. berghei zygotes and ookinetes from A. stephensi midgut content smears, 18 - 20 hours after the infective blood meal. Control mosquitoes, A to H; NeemAzal^® 50 mg/kg treated mosquitoes, I to T. See text for details.