Effect of oregano (<Emphasis Type="Italic">Origanum vulgare</Emphasis> L.) and thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> L.) essential oils on <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> (Protozoa: Kinetoplastida) growth and ultrastructure

In the present work, we have investigated the effect of essential oils obtained from Origanum vulgare L. (oregano) and Thymus vulgaris L. (thyme) on growth and ultrastructure of diverse evolutive forms of Trypanosoma cruzi. Culture epimastigotes and bloodstream trypomastigotes were incubated for 24 h with different concentrations of oregano or thyme essential oils and with thymol (the main constituent of thyme), and the inhibitory concentration (IC)₅₀ was determined by cell counting. Crude extract of oregano essential oil inhibited epimastigote growth (IC₅₀/24 h = 175 μg/ml) and also induced trypomastigote lysis (IC₅₀/24 h = 115 μg/ml). Thyme essential oil presented IC₅₀/24 h values of 77 μg/ml for epimastigotes and 38 μg/ml for trypomastigotes, while treatment with thymol resulted in an IC₅₀/24 h of 62 μg/ml for epimastigotes and 53 μg/ml for trypomastigotes. Scanning electron microscopy of treated cells showed few morphological alterations at the plasma membrane. Observation by transmission electron microscopy showed cytoplasmic swelling with occasional morphological alterations in plasma and flagellar membrane. Our data indicate that oregano and thyme essential oils are effective against T. cruzi, with higher activity of thyme, and that thymol may be the main component responsible for the trypanocidal activity.     

In vitro anthelmintic activity of the essential oils of <Emphasis Type="BoldItalic">Zanthoxylum zanthoxyloides</Emphasis> and <Emphasis Type="BoldItalic">Newbouldia laevis</Emphasis> against <Emphasis Type="BoldItalic">Strongyloides ratti</Emphasis>

The need for new anthelmintic with no chemical residues is becoming urgent. In a program aiming at the evaluation of plant as sources of new active molecules, the anthelmintic activities of the essential oils (EOs) obtained from either Zanthoxylum zanthoxyloides seeds or Newbouldia laevis leaves were evaluated against Strongyloides ratti by analyzing the results of two in vitro bioassays. These two plants and their tested parts were retained after an ethnopharmacology survey that confirmed their use by small-scale farmers for treatment of small ruminants affected by digestive helminths. The plants were harvested in Benin, and their EO were obtained by hydrodistillation. The EO yield of extraction was 0.65% (w/w) of for Z. zanthoxyloides seeds and 0.05% (w/w) for N. laevis. The chemical compositions of the two EOs were analyzed by gas chromatography coupled with mass spectrometry. The major constituents of the EO from Z. zanthoxyloides consisted of the following compounds: γ-terpinene (18 %), undecane (15 %), valencene (8.3 %), decanal (8.3 %), and 3-carene (6.7 %). In contrast, the major constituents of the EO from N. laevis leaves consisted of the following compounds: β-caryophyllene (36 %) and eugenol (5.8 %). An egg-hatching inhibition (EHI) assay was developed and a larval migration inhibition assay was used on S. ratti to examine the effects of the EOs and to evidence their inhibitory concentrations (IC₅₀ and IC₉₀) values on this nematode. Furthermore, the toxicity of the two EOs on Vero cell line was evaluated. When tested on S. ratti egg hatching, the two EOs resulted in similar IC₅₀ values (19.5 and 18.2 μg/ml for Z. zanthoxyloides and N. laevis, respectively), which were about sevenfold higher than that of the control (thiabendazole, IC₅₀ = 2.5 μg/ml). Larval migration was inhibited at similar concentrations for: Z. zanthoxyloides (IC₅₀ = 46 μg/ml), N. laevis (IC₅₀ = 51 μg/ml), and the control [levamisole (IC₅₀ = 36 μg/ml)]. No cytotoxicity was found on Vero cells because both EOs had IC₅₀ values higher than 50 μg/ml. Therefore, we have concluded that the EOs from two plants, used in folk medicine, may contain compounds with anthelmintic activity and could be used as improved traditional medicines or, at least, as food additives in a combined treatment for the control of helminth infections.     

Antileishmanial activity of crude extract and coumarin from <Emphasis Type="Italic">Calophyllum brasiliense</Emphasis> leaves against <Emphasis Type="Italic">Leishmania amazonensis</Emphasis>

Infections by protozoans of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials, which show renal and cardiac toxicity. As part of a search for new drugs against leishmaniasis, we evaluated the in vitro leishmanicidal activity of the (−) mammea A/BB. The compound (−) mammea A/BB is a coumarin-type mammea purified from a dichloromethane crude extract of leaves of Calophyllum brasiliense Cambess (Clusiaceae). The isolated compound was characterized using spectral analyses by UV, infrared, nuclear magnetic resonance of ¹H, ¹³C, distortionless enhancement by polarization transfer, correlation spectroscopy, heteronuclear multiple bond correlation, and heteronuclear multiple quantum coherence. The compound (−) mammea A/BB showed significant activity against promastigote and amastigote forms of L. amazonensis, with IC₅₀ (50% inhibition concentration of cell growth) at a concentration of 3.0 and 0.88 μg/ml and IC₉₀ (90% inhibition concentration of cell growth) of 5.0 and 2.3 μg/ml, respectively. The coumarin (−) mammea A/BB showed no cytotoxicity against J774G8 macrophages in culture, when it was tested at high concentrations that inhibited promastigote forms. Electron microscopy studies revealed considerable ultrastructural changes when promastigote forms of L. amazonensis were treated with 3.0 μg/ml of the coumarin (−) mammea A/BB for 72 h. We observed significant changes such as mitochondrial swelling with concentric membranes in the mitochondrial matrix and intense exocytic activity in the region of the flagellar pocket. Other alterations included the appearance of binucleate cells and multiple cytoplasmic vacuolization. These results showed that (−) mammea A/BB is a potent growth inhibitor of L. amazonensis and caused important changes in the parasite’s ultrastructure. This study provided new perspectives on the development of novel drugs with leishmanicidal activity obtained from natural products.     

Anti-leishmania activity of semi-purified fraction of <Emphasis Type="Italic">Jacaranda puberula</Emphasis> leaves

The crude methanolic extract from leaves of Jacaranda puberula showed activity against Leishmania (Leishmania) amazonensis. The extract presented active against promastigote forms with an inhibitory concentration 50% (IC₅₀) value of 88.0 μg/ml, but only moderated activity against amastigote forms; however in higher concentrations the extract showed cytotoxic effects. The bio-guided chromatographic fractionation the crude methanolic extract against amastigotes yielded a fraction with an IC₅₀ value of 14.0 μg/ml (without cytotoxic activity) in relation to the crude extract (IC₅₀ value, 359.0 μg/ml). These data indicate that J. puberula leaves contain active compounds, which should be further investigated for the development of new potential drugs against cutaneous leishmaniasis.     

Antileishmanial potential of a marine sponge, <Emphasis Type="Italic">Haliclona exigua</Emphasis> (Kirkpatrick) against experimental visceral leishmaniasis

In this study, we are reporting antileishmanial activity of a marine sponge Haliclona exigua, belonging to phylum Porifera. The crude methanol extract and its three fractions were tested both in vitro and in vivo. The crude extract exerted almost complete inhibition of promastigotes at 50 μg/ml and 76.4 ± 6.5% inhibition of intracellular amastigotes at 100 μg/ml concentration with IC₅₀ values of 18.6 μg/ml and 47.2 μg/ml, respectively. When administered to Leishmania donovani infected hamsters at a dose of 500 mg/kg × 5, p.o., it resulted in 72.2 ± 10.4% inhibition of intracellular amastigotes. At a lower dose (250 mg/kg), it exhibited 43.9 ± 5.1% inhibition. Among the fractions, highest antileishmanial activity both in vitro (>90%) and in vivo (60.9 ± 18.3%) was observed in n-butanol (soluble) fraction with IC₅₀ values of 8.2 μg/ml and 31.2 μg/ml against promastigotes and intracellular amastigotes, respectively. Hexane fraction also showed comparatively good activity against both the stages of parasites in vitro but was moderately active in leishmania-infected hamsters. Chloroform fraction resulted in 45 ± 10.2% inhibition in vivo at a dose of 500 mg/kg × 5, p.o., whereas it was inactive in vitro. n-Butanol (insoluble) fraction was inactive both in vitro and in vivo. Araguspongin C, an alkaloid isolated from n-butanol (soluble) fraction exhibited moderate inhibition of promastigotes and intracellular amastigotes at 100 μg/ml but showed weak antileishmanial action in vivo. Our findings indicate that this marine sponge has the potential to provide new lead toward development of an effective antileishmanial agent and, hence, calls for more exhaustive studies for exploiting the vast world of marine resources to combat the scourge of several parasitic diseases.     

In vitro and in vivo activity of <Emphasis Type="Italic">Aloe vera</Emphasis> leaf exudate in experimental visceral leishmaniasis

The leishmanicidal activity of Aloe vera leaf exudate (AVL) has been demonstrated in promastigotes and axenic amastigotes, but its effectiveness in animal models has not been evaluated. The presence of alkaloids, triterpenes, cyanidines, proanthocyanidines, tannins, and saponins in AVL was identified. Its effectiveness in four Leishmania donovani strains was studied both in promastigotes (IC₅₀ ranged from 70–115 μg/ml) and amastigotes (IC₅₀ ranged from 3.1–11.4 μg/ml). In amastigotes, the killing by AVL was facilitated through its induction of nitric oxide in leishmania-infected macrophages. The safety index was good as AVL up to 300 μg/ml remained non-toxic to monocytes and macrophages. In a L. donovani BALB/c mouse model, oral or subcutaneous administration of AVL (15 mg/kg body weight × 5 days) reduced parasitemia by >90% in the liver, spleen, and bone marrow without impairment of hepatic and renal functions. Collectively, we conclude that AVL shows promising antileishmanial activity and may provide a new lead agent in the treatment of Leishmaniasis. Chitra Mandal and Mitali Chatterjee should be considered as joint senior authors.     

Comparative studies of the anti-leishmanial activity of three <Emphasis Type="Italic">Crotalus durissus</Emphasis> ssp. venoms

In this study, we compared the anti-leishmanial activity of three crotalic venoms (Crotalus durissus terrificus–Cdt, Crotalus durissus cascavella–Cdca, and Crotalus durissus collilineatus–Cdcol). Different concentrations of each venom incubated with Leishmania (Leishmania) amazonensis promastigotes were used. Cdt venom exhibited a higher anti-leishmanial activity (Inhibitory concentration-IC50-value of 4.70 ± 1.72 μg/ml) in comparison with that of Cdca venom (IC50 value of 9.41 ± 1.21 μg/ml), while Cdcol venom increased parasite numbers in 50% at a concentration of 44.30 ± 2.18 μg/ml. In addition, this venom showed a low anti-leishmanial activity in higher concentrations (IC50 value of 281.00 ± 9.50 μg/ml). The main fractions of Cdca venom were isolated and assayed under similar conditions used for assessing crude venom. The most active fractions were gyroxin and crotamine that had IC50 values of 3.80 ± 0.52 μg/ml and 19.95 ± 4.21 μg/ml, respectively. Convulxin also inhibited parasite growth rate, although this effect was not dose-dependent. Crotoxin was the least effective fraction with an IC50 value of 99.80 ± 2.21 μg/ml. None of the protein fractions presented cytotoxic effects against J774 cells in culture. In vivo assays using BALB/c mice revealed that crotoxin and crotamine were the main toxic fractions. In conclusion, C. durissus cascavella venom has three main fractions with anti-leishmanial activity. These results open new possibilities to find proteins that might be used as possible agents against cutaneous leishmaniasis.     

Efficacy of the <Emphasis Type="Italic">Trichophyton ajelloi</Emphasis> and <Emphasis Type="Italic">Lagenidium giganteum</Emphasis> metabolites against mosquitoes after flash chromatography

Entomopathogens are significant natural enemies for mosquitoes. We have investigated the adulticidal efficacies of metabolites of Trichophyton ajelloi and Lagenidium giganteum against Culex quinquefasciatus and Aedes aegypti simultaneously. The T. ajelloi was grown on Sabouraud’s dextrose broth medium at 25 ± 2°C and relative humidity at 75 ± 5% for 15 days. L. giganteum was grown in peptone yeast extract glucose broth at 25 ± 2°C and relative humidity 75 ± 5% for 15 days. The filtrations of metabolites have been made by using Whatman-1 filter paper then with the flash chromatograph. The bioassays were conducted as per the World Health Organization’s methods and protocols (2006). In this significant investigations, the metabolites of T. ajelloi have been found highly susceptible against A. aegypti with LC₉₉-7.24 ml after an exposure time of 24 h with a comparison, the LC₉₉-66 ml was observed against C. quinquefasciatus after exposure of 24 h. Moreover, the L. giganteum metabolites have shown higher pathogenicity against C. quinquefasciatus with LC₉₉-11.3 ml and A. aegypti with LC₉₉-15.49 ml. Although, the efficacy in adults could be achieved with higher concentration can be significant also. Their adulticidal activities in different climatic zones are plausible with metabolites which have better LT values of T. ajelloi.     

Subcellular localization of an extracellular serine protease in<Emphasis Type="Italic"> Leishmania</Emphasis> (<Emphasis Type="Italic">Leishmania</Emphasis>)<Emphasis Type="Italic"> amazonensis</Emphasis>

Extracellular proteolytic activity was detected in a Leishmania (L.) amazonensis culture supernatant and a 56-kDa protein was purified using (NH₄)₂SO₄ precipitation followed by affinity chromatography on aprotinin–agarose. A rabbit serum obtained against the 56-kDa extracellular serine protease was used in order to analyze its location in L. (L.) amazonensis parasites. Immunocytochemistry studies revealed that the enzyme is mainly found in the flagellar pocket and cytoplasmic vesicles of promastigote forms, whereas in amastigotes, it is located in electron-dense structures resembling megasomes. These results indicate that the 56-kDa serine protease is released into the extracellular environment through the flagellar pocket; and its intracellular location suggests either a correlated enzymatic activity or intracellular trafficking.     

Differences in the in vitro susceptibility of mono-eukaryotic cultures of <Emphasis Type="Italic">Histomonas meleagridis,Tetratrichomonas gallinarum</Emphasis> and <Emphasis Type="Italic">Blastocystis</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> to natural organic compounds

Currently, all pharmaceuticals for the treatment or prophylaxis of blackhead disease (histomonosis) caused by the flagellate Histomonas meleagridis are banned from the market. Consequently, great interest exists on the finding of alternative drugs for the abatement of histomonosis. In this study, carvacrol, Cassia oil, an essential oil (EO) mixture containing thyme and rosemary EO and a Quillaja saponaria saponin were examined using in vitro assays for antiprotozoal and antibacterial activity testing established against cloned xenic cultures of different isolates of Histomonas meleagridis, Tetratrichomonas gallinarum and Blastocystis sp. Whereas similar minimal lethal concentrations (MLCs) of five Histomonas isolates were obtained for both carvacrol and the EO mixture as well as for the saponin, significantly different MLCs were observed for them with Cassia oil, ranging from 0.25 up to 0.50 μl/ml. Testing the Blastocystis isolates, different MLCs were obtained for all substances, whereas the Tetratrichomonas gallinarum isolates showed identical susceptibilities. The effects are independent of the bacteria, underlining the need of well-defined protozoan cultures for these investigations.     

Differential larvicidal efficacy of four species of <Emphasis Type="Italic">Vitex</Emphasis> against <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> larvae

The early fourth instar larvae of Culex quinquefasciatus, reared in the laboratory were used for larvicidal assay with leaf extracts of Vitex negundo, Vitex trifolia, Vitex peduncularis and Vitex altissima. The methanol extracts of the four species possessed varying levels of larvicidal nature. The highest larvicidal activity was found with the extract of V. trifolia (LC₅₀ = 41.41 ppm) followed by V. peduncularis (LC₅₀ = 76.28 ppm), V. altissima (LC₅₀ = 128.04 ppm) and V. negundo (LC₅₀ = 212.57 ppm).     

In vitro and in vivo efficacies of amlodipine against <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>

Listeria monocytogenes causes suppurative gastritis in BALB/c mice. We investigated the effect of the antihypertensive drug amlodipine (Aml) on the growth of L. monocytogenes in vitro and in vivo. Aml showed noteworthy inhibitory action (minimum inhibitory concentration, MIC₉₀ 32 μg/ml) against Listeria strains and demonstrated cidal (minimum bactericidal concentration, MBC 64 μg/ml) activity. Aml administered orally at 2.5 μg/g in female BALB/c mice for 7 days, commencing 4 days before oral challenge (1 × 10⁸ CFU/ml with L. monocytogenes ATCC 51774), significantly reduced bacterial counts in the stomach (P < 0.01), liver (P < 0.01), and spleen (P < 0.05), and decreased (P < 0.05) gastric lesions, neutrophilic infiltration, edema, vascular degeneration, and necrosis of gastric tissues. It caused the down-regulation of expression of inflammatory cytokines (IFN-γ, IL-1β, and TNF-α) compared to drug-free control. Aml may be used in the presence of an antibiotic as adjunct therapy that boosts the host immunity against Listeria. Further, QSAR studies might contribute in manipulating it as a lead compound for the synthesis of new, more effective non-antibiotics (helper compounds), perhaps devoid of side-effects, that could be recommended as compassionate therapy for listeriosis.     

The effect of phospholipase A2 from <Emphasis Type="Italic">Crotalus durissus collilineatus</Emphasis> on <Emphasis Type="Italic">Leishmania (Leishmania) amazonensis</Emphasis> infection

In this study, the effect of phospholipase A2 (PLA2) derived from Crotalus durissus collilineatus was evaluated in vitro and in vivo on experimental cutaneous leishmaniasis. The promastigote and amastigote forms treated with PLA2 presented increased growth rate. In vivo studies showed that PLA2-treated Leishmania (Leishmania) amazonensis promastigotes increased the size of lesions in BALB/c mice, and histopathological analysis showed numerous necrotic regions presenting a higher density of polymorphonuclear, mononuclear, and amastigote cells. Additionally, infected macrophages treated with PLA2 were able to generate prostaglandin E2 (PGE2). Cytokine quantification showed that the supernatant from infected macrophages presented moderate and high amounts of IL-2 and IL-10, respectively. However, in PLA2-treated infected macrophages, suppression of IL-2 levels occurred, but not of IL-10 levels. Observation also revealed that both the supernatant and lysate of L. (L.) amazonensis promastigotes exhibited PLA2 activity, which, in the presence of dexamethasone, showed no reduction in their activities; while glucocorticoid maintained the ability of promastigote forms to infect macrophages, which presented values similar to controls. In conclusion, the results indicate that PLA2 may be a progression factor for cutaneous leishmaniasis, since the PLA2 effect suppressed IL-2 levels and generated PGE2, an inflammatory lipid mediator.     

Larvicidal efficacy of <Emphasis Type="Italic">Jatropha curcas</Emphasis> and bacterial insecticide, <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>, against lymphatic filarial vector<Emphasis Type="Italic">, Culex quinquefasciatus</Emphasis> Say (Diptera: Culicidae)

The present study explored the effects of Jatropha curcas leaf extract and Bacillus thuringiensis israelensis larvicidal activity against the lymphatic filarial vector, Culex quinquefasciatus. Wights were selected for investigating the larvicidal potential against the first to fourth instar larvae of the laboratory-reared mosquito species, C. quinquefasciatus Say, in which the major lymphatic filariasis was used. The medicinal plants were collected from the area around Bharathiar University, Coimbatore. The dried plant materials were powdered by an electric blender. From the powder, 100 g of the plant materials was extracted with 300 ml of organic solvents of methanol for 8 h, using a Soxhlet apparatus, and filtered. The crude plant extracts were evaporated to dryness in a rotary vacuum evaporator. The plant extract showed larvicidal effects after 24 h of exposure; however, the highest larval mortality was found in the leaf extract of methanol J. curcas against the first to fourth instar larvae of values LC₅₀ = 1.200%, 1.290%, 1.358%, and 1.448% and LC₉₀ = 2.094%, 2.323%, 2.444%, and 2.544% and B. thuringiensis israelensis against the first to fourth instar larvae of values LC₅₀ = 9.332%, 9.832%, 10.212%, 10.622% and LC₉₀ = 15.225%, 15.508%, 15.887%, and 15.986% larvae of C. quinquefasciatus, respectively. No mortality was observed in the control. These results suggest methanol extracts of J. curcas and B. thuringiensis israelensis have potential to be used as an ideal eco-friendly approach for the control of the major lymphatic filarial vector, C. quinquefasciatus.     

Two new <Emphasis Type="Italic">Isospora</Emphasis> species from Brazilian tanager (<Emphasis Type="Italic">Ramphocelus bresilius dorsalis</Emphasis>) of South America

Two new coccidian (Protozoa: Apicomplexa: Eimeriidae) species from the Brazilian tanager Ramphocelus bresilius dorsalis are reported in the current study. Isospora cadimi n. sp. oocysts are spheroidal to sub-spheroidal, 24.2 × 22.9 μm, with a smooth and bi-layered wall, ∼1.1 μm. Micropyle, oocyst residuum, and polar granule are absent. Sporocysts are broadly ovoidal, 16.9 × 11.6 μm. Stieda and substieda bodies are present. Sporocyst residuum is present and sporozoites have refractile body and nucleus. Isospora navarroi n. sp. oocysts are spheroidal to sub-spheroidal, 21.4 × 20.6 μm, with a smooth and bi-layered wall, ∼1.1 μm. Micropyle, oocyst residuum, and polar granule are absent. Sporocysts are ellipsoidal, 16.1 × 10.2 μm. Stieda and substieda bodies are present. Sporocyst residuum is present and sporozoites have a robust posterior refractile body.     

Evaluation of larvicidal and nymphicidal potential of plant extracts against <Emphasis Type="Italic">Anopheles subpictus</Emphasis> Grassi, <Emphasis Type="Italic">Culex tritaeniorhynchus</Emphasis> Giles and <Emphasis Type="Italic">Aphis gossypii</Emphasis> Glover

The acetone, chloroform, ethyl acetate, hexane and methanol extracts of peel and leaf extracts of Citrus sinensis, Ocimum canum, Ocimum sanctum and Rhinacanthus nasutus were tested against fourth instar larvae of malaria vector, Anopheles subpictus Grassi, Japanese encephalitis vector, Culex tritaeniorhynchus Giles (Diptera: Culicidae) and feeding deterrence to nymphs of cotton pest, Aphis gossypii Glover (Homoptera: Aphididae). The larval and nymph mortality were observed after 24 h of exposure. All extracts showed moderate larvicidal and nymphicidal effects; however, the highest mortality was found in peel chloroform extract of C. sinensis, leaf ethyl acetate extracts of O. canum and O. sanctum and leaf chloroform extract of R. nasutus against the larvae of A. subpictus (LC₅₀ = 58.25, 88.15, 21.67 and 40.46 ppm; LC₉₀ = 298.31, 528.70, 98.34 and 267.20 ppm), peel methanol extract of C. sinensis, leaf methanol extract of O. canum, ethyl acetate extracts of O. sanctum and R. nasutus against the larvae of C. tritaeniorhynchus (LC₅₀ = 38.15, 72.40, 109.12 and 39.32 ppm; LC₉₀ = 184.67, 268.93, 646.62 and 176.39 ppm), peel hexane extract of C. sinensis, leaf methanol extracts of O. canum and R. nasutus and leaf ethyl acetate extract of O. sanctum against the nymph of A. gossypii (LC₅₀ = 162.89, 80.99, 73.27 and 130.19 ppm; LC₉₀ = 595.40, 293.33, 338.74 and 450.90 ppm), respectively. These results suggest that the peel methanol extracts of C. sinensis and O. canum, ethyl acetate leaf extract of O. sanctum and leaf chloroform and ethyl acetate extract of R. nasutus have the potential to be used as an ideal eco-friendly approach for the control of the A. subpictus, C. tritaeniorhynchus and A. gossypii.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

Characterization of <Emphasis Type="Italic">Taenia solium</Emphasis> cysticerci microsomal glutathione <Emphasis Type="Italic">S</Emphasis>-transferase activity

Glutathione S-transferase activity has been shown to be associated with the microsomal fraction of Taenia solium. Electron microscopy and subcellular enzyme markers indicate the purity of the microsomal fraction that contains the glutathione S-transferase activity. T. solium microsomes were solubilized under conditions used to solubilize integral microsomal proteins. This procedure proved necessary to obtain enzymatic activity. To characterize this parasite enzyme activity, several substrates and inhibitors were used. The optimum activity for microsomal glutathione S-transferase was found to be pH 6.6, with a specific enzyme activity of 0.9, 0.1, 0.067, 0.03, and 0.05 μmol min⁻¹ mg⁻¹ with the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 4-hydroxynonenal, 2,4-hexadienal, and trans-2-nonenal, respectively. No activity of glutathione peroxidase was observed. T. solium microsomes had an _app K _{m (GSH)} = 0.161 μM, _app K _{m (CDNB)} = 14.5 μM, and _app V _max of 0.15 and 27.9 μmol min⁻¹ mg⁻¹ for GSH and CDNB, respectively. T. solium microsomes were inhibited by several glutathione S-transferase enzyme inhibitors, and it was possible to establish a simple inhibition system as well as corresponding K _i ’s for each inhibitor. These results indicate that the T. solium microsomal glutathione S-transferase is different from the parasite cytoplasmic enzymes that catalyze similar reactions.     

In vitro antiplasmodial activity of ethanolic extracts of seaweed macroalgae against <Emphasis Type="Italic">Plasmodium falciparum</Emphasis>

Malaria is a major health problem in many developing countries. The drugs resistant Plasmodium falciparum causes the most virulent form of malaria in humans and it is described as a public health disaster causing increased morbidity and mortality. Thirteen seaweeds species which belong to four different families (Rhodomelaceae, Cladophoraceae, Ulvaceae, and Caulerpaceae) were collected from Mandapam coastal area and the seaweeds extracts were tested for in vitro antiplasmodial activity against P. falciparum. Among them, Caulerpa toxifolia (IC₅₀ 5.06 μg·ml⁻¹) showed potential antiplasmodial activity than other seaweeds extracts and it can be comparable with the positive control artemether (IC₅₀ 4.09 μg·ml⁻¹). Caulerpa peltata (IC₅₀ 16.69 μg·ml⁻¹) also exhibited good antiplasmodial activity and the IC₅₀ value is lesser than the positive control chloroquine (IC₅₀ 19.59 μg·ml⁻¹). Statistical analysis reveals that significant in vitro antiplasmodial activity (P < 0.05) was observed between the concentrations and time of exposure. The chemical injury to erythrocytes was also carried out and it shows that no morphological changes in erythrocytes by the ethanolic extract of seaweeds extracts after 48 h of incubation. The in vitro antiplasmodial activity might be due to the presence of sugars, proteins, and phenols in the ethanolic extracts of seaweeds. It is concluded from the present study that, the ethanolic extracts of seaweeds of C. toxifolia and C. peltata possesses lead compounds for development of antiplasmodial drugs.     

Drug-abbreviated infections of <Emphasis Type="Italic">Trichostrongylus colubriformis</Emphasis> and development of immunity in jirds (<Emphasis Type="Italic">Meriones unguiculatus</Emphasis>)

The objective of this study was to examine the development and the duration of immunity achieved with drug-abbreviated infections of Trichostrongylus colubriformis in jirds (Meriones unguiculatus). Jirds were primarily infected either by trickle infection with 6 × 100 infective larvae (L₃) of T. colubriformis at 3-day intervals or by a single infection with 600 L₃. On day 35 post-infection, one batch of jirds from each group was autopsied; the others were treated with oxfendazole at a dose of 5 mg/kg and were challenged with 1,000 L₃ on either day 7 or day 42 post-treatment. All jirds were autopsied at 17 days post-challenge. Trickle infection resulted in lower levels of egg production during the primary infection period. The systemic IgM and IgG antibody response was significantly stronger in trickle- and single-infected groups as compared with the negative control group (P < 0.01–P < 0.05). Significantly higher levels of intestinal IgA were demonstrated in trickle- and single-infected groups than in the negative control group (P < 0.01). Numbers of mucosal mast cells increased following infection, but this was not dependent on the type of immunisation. After challenge the extent of worm reduction was greater in trickle-infected than in single-infected subgroups. The IgM and IgG response was significantly stronger in challenged subgroups as compared with negative control subgroups (P < 0.01). However, the IgG response was weaker in control challenged subgroups than in challenged subgroups (P < 0.01). There was a negative correlation between the IgG response and the worm burden after the second challenge (r=−0.73). The acquired immunity to T. colubriformis infection in jirds developed within 5 weeks of primary infection. The level of immunity was higher after trickle infection than after single infection. Furthermore, the immunity persisted for at least 6 weeks after oxfendazole treatment in the absence of a worm burden and larval intake, which is very similar to the situation in domestic ruminant hosts. Received: 25 October1999 / Accepted: 23 December 1999