Antiplasmodial Bis-Indole Alkaloids from the Bark of Flindersia pimenteliana.

Three new (1: -3: ) and 2 known (4: -5: ) bis-indole alkaloids were identified from the bark of Flindersia pimenteliana (Rutaceae). The structures of 1: -3: were elucidated on the basis of their (+)-HRESESIMS and 2D NMR spectroscopic data. Antiplasmodial activity for 1: -3: against chloroquine sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum is also reported, with IC50 values ranging from 0.96 to 2.41 µg/mL. These results expand our knowledge of the structure-activity relationships of potently antiplasmodial isoborreverine-type alkaloids, the bioactivity of which have recently attracted significant attention in the literature.

Antiplasmodial ß-Triketone-Flavanone Hybrids from the Flowers of the Australian Tree Corymbia torelliana.

The methanol extract of the flowers of the Australian eucalypt tree Corymbia torelliana yielded six new ß-triketone-flavanone hybrids, torellianones A-F (1-6), the tetrahydroxycyclohexane torellianol A (7), and known ß-triketones (4 S)-ficifolidione (8) and (4 R)-ficifolidione (9), and ß-triketone-flavanones kunzeanone A (10) and kunzeanone B (11). Torellianones A and B, C and D, and E and F were each isolated as inseparable diastereomeric mixtures. Exchange correlations observed in a ROESY spectrum indicated that 5 and 6 also interconverted between stable conformers. The structures of 1-7 were elucidated from the analysis of 1D/2D NMR and MS data. Relative configurations of torellianones C-F and torrellianol A were determined from analysis of ROESY data. Compounds 1-10 were tested for antiplasmodial activity against a drug-sensitive (3D7) strain of Plasmodium falciparum, with 3-6 and 8-10 showing limited antiplasmodial activity, with IC50 values ranging from 3.2 to 16.6 µM.

HSQC-TOCSY Fingerprinting-Directed Discovery of Antiplasmodial Polyketides from the Marine Ascidian-Derived Streptomyces sp. (USC-16018).

Chemical investigations on the fermentation extract obtained from an ascidian-derived Streptomyces sp. (USC-16018) yielded a new ansamycin polyketide, herbimycin G (1), as well as a known macrocyclic polyketide, elaiophylin (2), and four known diketopiperazines (3⁻6). The structures of the compounds were elucidated based on 1D/2D NMR and MS data. The absolute configuration of 1 was established by comparison of experimental and predicted electronic circular dichroism (ECD) data. Antiplasmodial activities were tested for the natural products against chloroquine sensitive (3D7) and chloroquine resistant (Dd2) Plasmodium falciparum strains; the two polyketides (1⁻2) demonstrated an inhibition of >75% against both parasite strains and while 2 was highly cytotoxic, herbimycin G (1) showed no cytotoxicity and good predicted water solubility.

The need to compare: assessing the level of agreement of three high-throughput assays against Plasmodium falciparum mature gametocytes.

Whole-cell High-Throughput Screening (HTS) is a key tool for the discovery of much needed malaria transmission blocking drugs. Discrepancies in the reported outcomes from various HTS Plasmodium falciparum gametocytocidal assays hinder the direct comparison of data and ultimately the interpretation of the transmission blocking potential of hits. To dissect the underlying determinants of such discrepancies and assess the impact that assay-specific factors have on transmission-blocking predictivity, a 39-compound subset from the Medicines for Malaria Venture Malaria Box was tested in parallel against three distinct mature stage gametocytocidal assays, under strictly controlled parasitological, chemical, temporal and analytical conditions resembling the standard membrane feeding assay (SMFA). Apart from a few assay-specific outliers, which highlighted the value of utilizing multiple complementary approaches, good agreement was observed (average ΔpIC50 of 0.12 ± 0.01). Longer compound incubation times improved the ability of the least sensitive assay to detect actives by 2-fold. Finally, combining the number of actives identified by any single assay with those obtained at longer incubation times yielded greatly improved outcomes and agreement with SMFA. Screening compounds using extended incubation times and using multiple in vitro assay technologies are valid approaches for the efficient identification of biologically relevant malaria transmission blocking hits.

In vitro and ex vivo activity of an Azadirachta indica A.Juss. seed kernel extract on early sporogonic development of Plasmodium in comparison with azadirachtin A, its most abundant constituent.

BACKGROUND: NeemAzal® (NA) is a quantified extract from seed kernels of neem, Azadirachta indica A.Juss. (Meliaceae), with a wide spectrum of biological properties, classically ascribed to its limonoid content. NA contains several azadirachtins (A to L), azadirachtin A (AzaA) being its main constituent. AzaA has been shown to inhibit microgamete formation of the rodent malaria parasite Plasmodium berghei, and NA was found to completely inhibit the transmission of Plasmodium berghei to Anopheles stephensi mosquitoes when administered to gametocytemic mice at a corresponding AzaA dose of 50mg/kg before exposure to mosquitoes. PURPOSE: The present study was aimed at i) assessing the pharmacodynamics and duration of action of NA and AzaA against P. berghei exflagellation in systemic circulation in mice and ii) elucidating the transmission blocking activity (TBA) of the main NA constituents. STUDY DESIGN: The NA and AzaA pharmacodynamics on exflagellation were assessed through ex vivo exflagellation assays, while TBA of NA constituents was evaluated through in vitro ookinete development assay. METHODS: Pharmacodynamics experiments: Peripheral blood from P. berghei infected BALB/c mice with circulating mature gametocytes, were treated i.p. with 50mg/kg and 100mg/kg pure AzaA and with NeemAzal® (Trifolio-M GmbH) at the corresponding AzaA concentrations. The effect magnitude and duration of action of compounds was estimated by counting exflagellation centers, formed by microgametocytes in process of releasing flagellated gametes, at various time points after treatment in ex vivo exflagellation tests. Ookinete Development Assay: The direct effects of NeemAzal® and AzaA on ookinete development were measured by fluorescence microscopy after incubation of gametocytemic blood with various concentrations of test substances in microplates for 24h. RESULTS: The exflagellation tests revealed an half-life of NA anti-plasmodial compounds of up to 7h at a NA dose corresponding to 100mg/kg equivalent dose of AzaA. The ookinete development assay showed an increased activity of NA against early sporogonic stages compared to that of AzaA. The IC50 value determined for NA was 6.8µg/ml (CI95: 5.95-7.86), about half of the AzaA IC50 (12.4µg/ml; CI95: 11.0-14.04). CONCLUSION: The stronger activity of NA, when compared to AzaA, could not be explained by an additive or synergistic effect by other azadirachtins (B, D and I) present in NA. In fact, the addition of these compounds at 50µM concentration to AzaA did not evidence any decrease of the IC50 against early sporogonic stages to that obtained with AzaA alone. It is likely that other non-limonoid compounds present in NA may contribute to AzaA activity and enhanced pharmacodynamics against exflagellation both in vitro and in vivo.

Transmission blocking effects of neem (Azadirachta indica) seed kernel limonoids on Plasmodium berghei early sporogonic development.

Azadirachta indica, known as neem tree and traditionally called "nature's drug store" makes part of several African pharmacopeias and is widely used for the preparation of homemade remedies and commercial preparations against various illnesses, including malaria. Employing a bio-guided fractionation approach, molecules obtained from A. indica ripe and green fruit kernels were tested for activity against early sporogonic stages of Plasmodium berghei, the parasite stages that develop in the mosquito mid gut after an infective blood meal. The limonoid deacetylnimbin (3) was identified as one the most active compounds of the extract, with a considerably higher activity compared to that of the close analogue nimbin (2). Pure deacetylnimbin (3) appeared to interfere with transmissible Plasmodium stages at a similar potency as azadirachtin A. Considering its higher thermal and chemical stability, deacetylnimbin could represent a suitable alternative to azadirachtin A for the preparation of transmission blocking antimalarials.

A simple and predictive phenotypic High Content Imaging assay for Plasmodium falciparum mature gametocytes to identify malaria transmission blocking compounds.

Plasmodium falciparum gametocytes, specifically the mature stages, are the only malaria parasite stage in humans transmissible to the mosquito vector. Anti-malarial drugs capable of killing these forms are considered essential for the eradication of malaria and tools allowing the screening of large compound libraries with high predictive power are needed to identify new candidates. As gametocytes are not a replicative stage it is difficult to apply the same drug screening methods used for asexual stages. Here we propose an assay, based on high content imaging, combining "classic" gametocyte viability readout based on gametocyte counts with a functional viability readout, based on gametocyte activation and the discrimination of the typical gamete spherical morphology. This simple and rapid assay has been miniaturized to a 384-well format using acridine orange staining of wild type P. falciparum 3D7A sexual forms, and was validated by screening reference antimalarial drugs and the MMV Malaria Box. The assay demonstrated excellent robustness and ability to identify quality hits with high likelihood of confirmation of transmission reducing activity in subsequent mosquito membrane feeding assays.

Plasmodium transmission blocking activities of Vernonia amygdalina extracts and isolated compounds.

BACKGROUND: Medicinal plants are a validated source for discovery of new leads and standardized herbal medicines. The aim of this study was to assess the activity of Vernonia amygdalina leaf extracts and isolated compounds against gametocytes and sporogonic stages of Plasmodium berghei and to validate the findings on field isolates of Plasmodium falciparum. METHODS: Aqueous (Ver-H2O) and ethanolic (Ver-EtOH) leaf extracts were tested in vivo for activity against sexual and asexual blood stage P. berghei parasites. In vivo transmission blocking effects of Ver-EtOH and Ver-H2O were estimated by assessing P. berghei oocyst prevalence and density in Anopheles stephensi mosquitoes. Activity targeting early sporogonic stages (ESS), namely gametes, zygotes and ookinetes was assessed in vitro using P. berghei CTRPp.GFP strain. Bioassay guided fractionation was performed to characterize V. amygdalina fractions and molecules for anti-ESS activity. Fractions active against ESS of the murine parasite were tested for ex vivo transmission blocking activity on P. falciparum field isolates. Cytotoxic effects of extracts and isolated compounds vernolide and vernodalol were evaluated on the human cell lines HCT116 and EA.hy926. RESULTS: Ver-H2O reduced the P. berghei macrogametocyte density in mice by about 50% and Ver-EtOH reduced P. berghei oocyst prevalence and density by 27 and 90%, respectively, in An. stephensi mosquitoes. Ver-EtOH inhibited almost completely (>90%) ESS development in vitro at 50 µg/mL. At this concentration, four fractions obtained from the ethylacetate phase of the methanol extract displayed inhibitory activity >90% against ESS. Three tested fractions were also found active against field isolates of the human parasite P. falciparum, reducing oocyst prevalence in Anopheles coluzzii mosquitoes to one-half and oocyst density to one-fourth of controls. The molecules and fractions displayed considerable cytotoxicity on the two tested cell-lines. CONCLUSIONS: Vernonia amygdalina leaves contain molecules affecting multiple stages of Plasmodium, evidencing its potential for drug discovery. Chemical modification of the identified hit molecules, in particular vernodalol, could generate a library of druggable sesquiterpene lactones. The development of a multistage phytomedicine designed as preventive treatment to complement existing malaria control tools appears a challenging but feasible goal.

Impact of repeated NeemAzal-treated blood meals on the fitness of Anopheles stephensi mosquitoes.

BACKGROUND: Herbal remedies are widely used in many malaria endemic countries to treat patients, in particular in the absence of anti-malarial drugs and in some settings to prevent the disease. Herbal medicines may be specifically designed for prophylaxis and/or for blocking malaria transmission to benefit both, the individual consumer and the community at large. Neem represents a good candidate for this purpose due to its inhibitory effects on the parasite stages that cause the clinical manifestations of malaria and on those responsible for infection in the vector. Furthermore, neem secondary metabolites have been shown to interfere with various physiological processes in insect vectors. This study was undertaken to assess the impact of the standardised neem extract NeemAzal on the fitness of the malaria vector Anopheles stephensi following repeated exposure to the product through consecutive blood meals on treated mice. METHODS: Batches of An. stephensi mosquitoes were offered 5 consecutive blood meals on female BALB/c mice treated with NeemAzal at an azadirachtin A concentration of 60, 105 or 150 mg/kg. The blood feeding capacity was estimated by measuring the haematin content of the rectal fluid excreted by the mosquitoes during feeding. The number of eggs laid was estimated by image analysis and their hatchability assessed by direct observations. RESULTS: A dose and frequency dependent impact of NeemAzal treatment on the mosquito feeding capacity, oviposition and egg hatchability was demonstrated. In the 150 mg/kg treatment group, the mosquito feeding capacity was reduced by 50% already at the second blood meal and by 50 to 80% in all treatment groups at the fifth blood meal. Consequently, a 50 - 65% reduction in the number of eggs laid per female mosquito was observed after the fifth blood meal in all treatment groups. Similarly, after the fifth treated blood meal exposure, hatchability was found to be reduced by 62% and 70% in the 105 and 150 mg/kg group respectively. CONCLUSIONS: The findings of this study, taken together with the accumulated knowledge on neem open the challenging prospects of designing neem-based formulations as multi-target phytomedicines exhibiting preventive, parasite transmission-blocking as well as anti-vectorial properties.

Transmission blocking activity of Azadirachta indica and Guiera senegalensis extracts on the sporogonic development of Plasmodium falciparum field isolates in Anopheles coluzzii mosquitoes.

BACKGROUND: Targeting the stages of the malaria parasites responsible for transmission from the human host to the mosquito vector is a key pharmacological strategy for malaria control. Research efforts to identify compounds that are active against these stages have significantly increased in recent years. However, at present, only two drugs are available, namely primaquine and artesunate, which reportedly act on late stage gametocytes. METHODS: In this study, we assessed the antiplasmodial effects of 5 extracts obtained from the neem tree Azadirachta indica and Guiera senegalensis against the early vector stages of Plasmodium falciparum, using field isolates. In an ex vivo assay gametocytaemic blood was supplemented with the plant extracts and offered to Anopheles coluzzii females by membrane feeding. Transmission blocking activity was evaluated by assessing oocyst prevalence and density on the mosquito midguts. RESULTS: Initial screening of the 5 plant extracts at 250 ppm revealed transmission blocking activity in two neem preparations. Up to a concentration of 70 ppm the commercial extract NeemAzal completely blocked transmission and at 60 ppm mosquitoes of 4 out of 5 replicate groups remained uninfected. Mosquitoes fed on the ethyl acetate phase of neem leaves at 250 ppm showed a reduction in oocyst prevalence of 59.0% (CI95 12.0 - 79.0; p < 10-4) and in oocyst density of 90.5% (CI95 86.0 - 93.5; p < 10-4 ), while the ethanol extract from the same plant part did not exhibit any activity. No evidence of transmission blocking activity was found using G. senegalensis ethyl acetate extract from stem galls. CONCLUSIONS: The results of this study highlight the potential of antimalarial plants for the discovery of novel transmission blocking molecules, and open up the potential of developing standardized transmission blocking herbal formulations as malaria control tools to complement currently used antimalarial drugs and combination treatments.

Whole-cell in vitro screening for gametocytocidal compounds.

The discovery of new chemical starting points with the ability to inhibit Plasmodium falciparum sexual stages, and therefore block the disease transmission, is urgently required. These will form the basis for the development of new therapeutic combinations for the treatment and elimination of malaria and the ultimate goal of global eradication. Recent screening of large chemical libraries against the parasite asexual stages has resulted in the public availability of focused subsets of known antimalarial actives, which represent an excellent starting point for the identification of new gametocytocidal compounds. New stage-specific methodologies aimed at increasing the throughput for assessing compound activity against in vitro cultured gametocytes have recently been published. This article discusses the challenges of assay-oriented large-scale gametocyte culturing and reviews the state-of-the art in gametocytocidal assay development and outcomes.

Antimalarial plant remedies from Burkina Faso: their potential for prophylactic use.

ETHNOPHARMACOLOGICAL RELEVANCE: Saye, a combination remedy prepared from Cochlospermum planchonii Hook.f. (Cochlospermaceae), Cassia alata L. (Fabaceae) and Phyllanthus amarus Schumach. et Thonn. (Euphorbiaceae), N'Dribala, a Cochlospermum planchonii root decoction, and a fruit preparation of Azadirachta indica A. Juss. (Meliaceae) are plant remedies of the folk medicine in Burkina Faso and are commonly used by traditional healers for the treatment of malaria. AIM OF THE STUDY: This study aimed at validating the antiplasmodial activity of the preparations and at estimating their potential for prophylaxis, using the murine malaria system Plasmodium berghei/Anopheles stephensi. MATERIALS AND METHODS: Aqueous extracts were orally administered to mice (6 animals per treatment group) at a daily dose of 200mg/kg body weight for nine days, applying protocols that mimic as much as possible traditional recipes and treatment schemes. RESULTS: Saye, N'Dribala and Azadirachta indica preparations revealed prophylactic activity, reducing parasitaemia in treated mice, with respect to controls, by 52.0% (CI(95) 46.1-57.9), 45.5% (CI(95) 44.5-46.5) and 45.0% (CI(95) 41.1-48.9), respectively. No evidence of transmission blocking effects was detected with any of the tested remedies. CONCLUSIONS: This study confirms, in the murine malaria system, the antiplasmodial properties of the examined remedies on the Plasmodium stages developing in the vertebrate host, thus encouraging studies aiming at identifying the active fractions and compounds responsible for the described activity and to develop standardized prophylactic remedies.

Potential of a Khaya ivorensis -Alstonia boonei extract combination as antimalarial prophylactic remedy.

ETHNOPHARMACOLOGICAL RELEVANCE: The decoction of the combined stem barks of Khaya ivorensis A. Chev. (Meliaceae) and Alstonia boonei De Wild (Apocynaceae) has a history of use in traditional medicine of central Cameroon for malaria treatment but also for the prevention of the disease. AIM OF THE STUDY: The purpose of this investigation was to determine the antiplasmodial activity of Khaya ivorensis (K) and Alstonia boonei (A) preparations in the murine malaria model Plasmodium berghei/Anopheles stephensi, to estimate their prophylactic potential and to assess acute and sub-acute toxicity of the formulations prepared according to the traditional recipes. MATERIALS AND METHODS: Aqueous extracts from the stem-bark of the two plants were prepared and tested separately and in combination. BALB/c mice were treated for 9 days and challenged on day 3 by exposure to mosquitoes infected with Plasmodium berghei. Treatment doses ranged between 200 and 400mg/kg/day, corresponding approximately to the dosage applied by traditional healers to cure malaria patients or prevent the disease. Parasitemia reduction in treated animals was calculated from Giemsa smear counts, of two replicate experiments. To estimate acute toxicity in terms of median lethal dose (LD50), geometrically increasing doses were administered to mice. Sub-acute toxicity of the herbal combination (KA) was investigated by administering the same doses as in the antiplasmodial activity test for a period of 14 days, followed by 14 days of recovery observation. Locomotor activity (Open Field Test), body weight, liver and kidney morphology were monitored. RESULTS: The combination KA was found to exhibit antiplasmodial activity in the murine malaria model. In mice treated with the combination remedy at a dosage of 200mg/kg/day, parasitemia values of 6.2% ± 1.7 and 6.5% ± 0.8 were recorded, compared to 10.8% ± 1.3 and 12.0% ± 4.0 in controls (p<0.01). Doubling the dosage of the extracts did not significantly increase parasite suppression. When extracts of K and A were administered separately at a dosage of 400mg/kg, a reduction in parasitemia was still obtained, but it did not reach statistical significance. Toxicity studies yielded comforting results: the LD50 was estimated to be greater than 2779.5mg/kg. Moreover, mice exposed to the fourteen-day repeated-dose toxicity test (sub-acute toxicity test) did not display weight loss, liver or kidney morphological modifications, significant alterations in locomotor activity or any other sign of illness. CONCLUSION: The antiplasmodial activity and the wide dose interval between the therapeutic dosage and the toxic dosage exhibited by the KA herbal combination in the murine malaria model argue in favor of its use as an antimalarial prophylactic remedy. It remains to be demonstrated by human clinical trials whether the combination remedy, when taken by inhabitants during malaria transmission season, can reduce parasite density and lead to a reduction of malaria episodes in the community.

Antiplasmodial triterpenoids from the fruits of neem, Azadirachta indica.

Eight known and two new triterpenoid derivatives, neemfruitins A (9) and B (10), have been isolated from the fruits of neem, Azadirachta indica, a traditional antimalarial plant used by Asian and African populations. In vitro antiplasmodial tests evidenced a significant activity of the known gedunin and azadirone and the new neemfruitin A and provided useful information about the structure-antimalarial activity relationships in the limonoid class.

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

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.

Antiplasmodial activity of Punica granatum L. fruit rind.

AIM OF THE STUDY: Sun-dried rind of the immature fruit of Punica granatum L. (Punicaceae) (Pg) is presently used as a herbal formulation (OMARIA) in Orissa, India, for the therapy and prophylaxis of malaria. The aims of this study were (i) to assess in vitro the antiplasmodial activity of the methanolic extract, of a tannin enriched fraction and of compounds/metabolites of the antimalarial plant, (ii) to estimate the curative efficacy of the Pg extracts and (iii) to explore the mechanism of action of the antiplasmodial compounds. Urolithins, the ellagitannin metabolites, were also investigated for antiplasmodial activity. MATERIALS AND METHODS: Chloroquine-susceptible (D10) and -resistant (W2) strains of Pf were used for in vitro studies and the rodent malaria model Plasmodium berghei-BALB/c mice was used for in vivo assessments. Recombinant plasmepsins 2 and 4 were used to investigate the interference of Pg compounds with the metabolism of haemoglobin by malaria parasites. RESULTS: The Pg methanolic extract (Pg-MeOH) inhibited parasite growth in vitro with a IC(50) of 4.5 and 2.8 microg/ml, for D10 and W2 strain, respectively. The activity was found to be associated to the fraction enriched with tannins (Pg-FET, IC(50) 2.9 and 1.5 microg/ml) in which punicalagins (29.1%), punicalins, ellagic acid (13.4%) and its glycoside could be identified. Plasmepsin 2 was inhibited by Pg-MeOH extract and by Pg-FET (IC(50) 7.3 and 3.0 microg/ml), which could partly explain the antiparasitic effect. On the contrary, urolithins were inactive. Both Pg-MeOH extract and Pg-FET did not show any in vivo efficacy in the murine model. CONCLUSIONS: The in vitro studies support the use of Pg as antimalarial remedy. Possible explanations for the negative in vivo results are discussed.

Impact of the botanical insecticide Neem Azal on survival and reproduction of the biting louse Damalinia limbata on angora goats.

Secondary metabolites present in the neem tree (Azadirachta indica A. Juss, Meliaceae), exhibit a wide range of biological activities in insects. However, few studies have been undertaken to assess the potential of neem products as insecticides for the control of ectoparasites of domestic animals. This study was undertaken to estimate the efficacy of Neem Azal, an azadirachtin-rich extract of neem seeds, in controlling Damalinia limbata (Phthiraptera) louse infestation of angora goats. The study was conducted on a fibre animal farm situated in Central Italy. Groups of 11-12 goats were treated with Neem Azal at an azadirachtin concentration of 650ppm or 125ppm, with Neguvon or were left untreated. Their louse burden was assessed fortnightly to monthly for 22 weeks. A reduction in louse densities of 76-96% was observed from week 2 to week 18 after treatment with the neem solution containing azadirachtin at a concentration of 650ppm. At the lower test concentration (125ppm) a reduction of 60-92% could be recorded from week 2 to week 14. Neem Azal was found to reduce the survival of both adult and nymph stages of D. limbata and to interfere with oviposition and oogenesis of female lice. A decrease in oviposition was observed in neem exposed female lice and the examination of their ovaries revealed morphological alterations in both vitellogenic and previtellogenic ovarioles at the follicular and germinal level. Since neem compounds target different life stages and physiological processes of D. limbata, the development of insecticide resistance by biting lice exposed to neem-based insecticides appears unlikely. For this reason and for its prolonged activity, which in principle allows angora goats to be protected for a large part of the mohair production cycle, neem-based insecticides may have a potential interest for mohair producing breeders.