Lethal and repellent effect of amitraz, eugenol and thymol against Triatoma infestans, the main vector of Trypanosoma cruzi in the southern of America.

The lethal and repellent effect of the synthetic insecticide amitraz and the botanical insecticides eugenol and thymol separately and together in binary mixtures was tested against late-stage nymphs of a susceptible strain of Triatoma infestans, the main vector of Trypanosoma cruzi, the etiological agent of Chagas disease, in the Southern Cone of America. For the lethality study, the LD50 was determined for each insecticide alone and in binary mixture by topical application. The combination index (CI) was established to quantify interactions occurring between the insecticides. The repellent effect was tested using the area preference technique. The lethal effect of amitraz was 11 and 34 times more potent than that of thymol and eugenol, respectively. Only the combination of eugenol and amitraz at high concentrations showed a synergistic effect (CI: 0.3). The repellent activity of monoterpenes after 30 min of exposure was significant at 780 and 78 µg/cm2 for eugenol and thymol, respectively. The residual repellent effect of eugenol lasted for one week at the concentrations of 1170 and 1560 µg/cm2 , whereas thymol managed to retain its repellent effect for two weeks at concentrations of 1560 and 3900 µg/cm2 .

Current status of resistance to ivermectin in Rhipicephalus sanguineus sensu stricto infesting dogs in three provinces in Argentina.

Intensive use of macrocyclic lactones for parasite control exerts strong selective pressure for arthropods such as ticks to become resistant to them. Rhipicephalus sanguineus sensu stricto is a tick and disease vector of significant public health and veterinary importance worldwide. We assessed the toxicological response to the macrocyclic lactone ivermectin (IVM) in R. sanguineus s.s. infesting dogs in Argentina. Samples of nine tick populations were obtained by inspecting dogs at veterinary clinics, hospitals, or rural areas in the provinces of San Luis, Rio Negro, and Buenos Aires. Pet owners were interviewed to gather data on the history of dog treatment with ectoparasiticides. The larval immersion test was used to assess the toxicological response of R. sanguineus s.s. to IVM. Dose-response mortality regressions, lethal concentrations (LC), and slope were calculated by probit analysis. The lowest LC concentrations were used to designate the reference susceptible population because a laboratory reference strain of R. sanguineus s.s. does not exist in Argentina. Compared with the most susceptible tick population in this study, six populations (66.66%) were classified as resistant to IVM. A clear interpopulation variation in the level of IVM resistance was present (resistance ratios at LC50% ranged from 1.0 to 18.33 and at LC99% ranged from 1.0 to 8.96). In San Luis Province, all tick populations were classified as resistant. The highest level of IVM resistance (resistance ratio at LC50%:18.83 and LC99%:8.96) was found in a population of R. sanguineus s.s. from a rural area in the province of Buenos Aires. It is concluded that populations of R. sanguineus s.s. from dogs in three provinces of Argentina were resistant to IVM. Clear interpopulation variation in the level of IVM resistance was present.

A study of the effects of imidacloprid under laboratory and field conditions on nymphs of Triatoma infestans (Hemiptera: Reduviidae).

The aim of this study was to determine imidacloprid's lethal activity against fifth-instar nymphs of Triatoma infestans. In the first stage of this work, it was assayed the topical application of this insecticide on non-fed and repletion-fed nymphs. Results showed a DL50 three times greater in non-fed bugs than in those fully engorged. The presence of food determined less time for the insecticide's maximum lethal effect: 24 h post topical application in fed nymphs and 72 h in non-fed nymphs. In the study's second stage, we assayed a xenointoxication assay on dogs. The commercial products, Advantage®, Bayer (imidacloprid 10 % p/v) and Power Ultra®, Brouwer (imidacloprid 5.15 %, permethrin 40 % and piperonyl butoxide [PBO] 3%) were evaluated. Following administration of the insecticide, nymphs were fed on dogs 24, 72, 168, 240 and 336 h. Blood intake was similar in nymphs exposed to treated dogs versus controls. Although both commercial products showed low triatomicidal activity, a higher efficacy of the product combining imidacloprid with the synergist piperonyl butoxide and permethrin versus the product with imidacloprid as the only active ingredient was observed, causing in nymphs a mortality rate of 36.3 % and 20.7 %, respectively. Our results suggest that imidacloprid, alone or in combination with permethrin and PBO, is not an alternative for control of T. infestans.

Repellent and Lethal Activities of Extracts From Fruits of Chinaberry (Melia azedarach L., Meliaceae) Against Triatoma infestans.

Triatoma infestans is the principal vector of Trypanosoma cruzi, parasite responsible of Chagas's Disease transmission in Argentina. Pyrethroids have become common pesticides for the control of T. infestans but increasing resistance encourages the search of new alternatives and the use of natural products for biological control arises as a new strategy. Melia azedarach L. is originated from the Himalaya's region and several compounds are part of its rich phytochemistry. Folk medicine of the plant is due to its repellent and insecticidal activities. Aims of this work were to evaluate the repellent activity of methanolic and acetonic extracts from fruits of M. azedarach by means of the area preference method of fifth and first nymph stages as well as to test the acute lethal effect of the more repellent extract by means of direct application on cuticle on both stages. For repellence, qualitative filter papers were divided into two halves, one treated with methanolic (ME) or acetonic (AC) extract and the other without treatment. Controls were impregnated half with methanol or acetone and half without the solvents. One nymph was located in each Petri or well and repellence percentage was determined. For the lethal effect, fasted and fed to repletion 5th stage nymphs were topically administered with different concentrations of AC and deaths were registered after 24, 48, 72, 96, and 120 h. Phytochemical analysis of extracts was performed as well. AC demonstrated high repellent activity (100%, both stages), whereas ME extract activity was slight (10-21%). AC extract was selected for lethal assays due to early repellent activity. Fed to repletion nymphs were more sensitive to the lethal activity of the extract when compared to fasted nymphs (LD50: 11.5 vs. 23.1 µg/insect, respectively). Phytochemistry assays of extracts showed a higher concentration of flavonoids, alkaloids and triterpenes for AC. Considering these results, next assays will include the test of Melia azedarach extract on T. infestans that are resistant to pyrethroids for a possible synergism between AC and the pesticides.

Evaluation of the toxic effects of doramectin, ivermectin and eprinomectin against Triatoma infestans using a rat model.

INTRODUCTION: Pyrethroids have been frequently and intensively used for controlling the triatomine vectors of Trypanosoma cruzi. The emergence of resistance to these insecticides has resulted in an urgent need to identify novel, alternative and complementary control strategies. OBJECTIVE: To evaluate the toxic effects of ivermectin, doramectin and eprinomectin on the bloodfeeding behaviour of Triatoma infestans using a rodent model. MATERIALS AND METHODS: Fifth instar nymphs of T. infestans were fed at different times on Wistar rats pretreated with doramectin, ivermectin, eprinomectin or dimethylsulfoxide (excipient control) topically or orally administered. We determined the effects of each insecticide and of dimethyl sulfoxide on the amount of ingested blood, the volume of faecal discharge, and the mortality rates in triatomines. RESULTS: Only the rate of triatomine mortality was associated with the antiparasitic compounds administered and the route of administration utilized. Doramectin administration was associated with a higher mortality rate (21.5%) than ivermectin, eprinomectin and dimethylsulfoxide (16, 11 and 2.5%, respectively), and topical administration was found to be most effective for inducing mortality (23 vs. 9.3 %). CONCLUSION: These results demonstrate the toxic effects of the three assessed insecticides on T. infestans. The administration of ecto/endoparasiticides to domiciliary or peridomiciliary animals may serve as an interesting complementary strategy to the use of pyrethroids for the control of T. infestans.

Evaluación del efecto tóxico de la doramectina, la ivermectina y la eprinomectina sobre Triatoma infestans en un modelo de rata / Evaluation of the toxic effects of doramectin, ivermectin and eprinomectin against Triatoma infestans using a rat model

Resumen Introducción. La principal herramienta para el control de los triatominos, vectores de Trypanosoma cruzi, ha sido el uso masivo e intensivo de piretroides. La aparición de resistencia a estas moléculas ha planteado la necesidad de encontrar estrategias nuevas, alternativas y complementarias de control. Objetivo. Evaluar el efecto tóxico de la ivermectina, la doramectina y la eprinomectina sobre Triatoma infestans y sus consecuencias en la alimentación con sangre en un modelo de roedor. Materiales y métodos. Se alimentaron ninfas de quinto estadio de T. infestans en distintos momentos sobre ratas Wistar tratadas previamente con doramectina, ivermectina, eprinomectina o dimetilsulfóxido (excipiente de control), administrados tópicamente o por vía oral. Se determinó el efecto de cada endectocida y del dimeltilsulfóxido en la cantidad de sangre ingerida, el volumen de excreciones y el porcentaje de mortalidad. Resultados. Únicamente la mortalidad de los insectos dependió del endectocida suministrado a las ratas y de la vía de administración utilizada. La doramectina causó mayor mortalidad (21,5 %) comparada con la ivermectina, la eprinomectina y el dimetilsulfóxido (16, 11 y 2,5 %, respectivamente), y la administración tópica fue más efectiva que la vía oral (23 Vs. 9,3 %). Conclusión. Los resultados obtenidos demuestran el efecto tóxico de los tres endectocidas en T. infestans. Su utilización en animales domiciliarios o que viven en el peridomicilio podría ser una interesante estrategia complementaria de la aspersión con piretroides para el control de T. infestans.

Abstract Introduction: Pyrethroids have been frequently and intensively used for controlling the triatomine vectors of Trypanosoma cruzi. The emergence of resistance to these insecticides has resulted in an urgent need to identify novel, alternative and complementary control strategies. Objective: To evaluate the toxic effects of ivermectin, doramectin and eprinomectin on the blood-feeding behaviour of Triatoma infestans using a rodent model. Materials and methods: Fifth instar nymphs of T. infestans were fed at different times on Wistar rats pretreated with doramectin, ivermectin, eprinomectin or dimethylsulfoxide (excipient control) topically or orally administered. We determined the effects of each insecticide and of dimethyl sulfoxide on the amount of ingested blood, the volume of faecal discharge, and the mortality rates in triatomines. Results: Only the rate of triatomine mortality was associated with the antiparasitic compounds administered and the route of administration utilized. Doramectin administration was associated with a higher mortality rate (21.5%) than ivermectin, eprinomectin and dimethylsulfoxide (16, 11 and 2.5%, respectively), and topical administration was found to be most effective for inducing mortality (23 vs. 9.3 %). Conclusion: These results demonstrate the toxic effects of the three assessed insecticides onT. infestans. The administration of ecto/endoparasiticides to domiciliary or peridomiciliary animals may serve as an interesting complementary strategy to the use of pyrethroids for the control of T. infestans.

Apoptotic activity of isoespintanol derivatives in human polymorphonuclear cells / Actividad apoptótica de derivados semisintéticos del isopentanol en células polimorfonucleares humanas

Background: Inflammation is a complex physiopathologic response to different stimuli. Recently, some pharmacological strategies have been proposed that could be used for resolution of inflammation by enhancing apoptosis of inflammatory cells. Objectives: To study in vitro apoptotic activity of isoespintanol [ISO] and of two semi-synthetic derivatives, bromide isoespintanol [BrI] and demethylated isoespintanol [DMI], in human polymorphonuclear (PMN) cells. Methods: PMN were exposed to the different concentrations of ISO, BrI and DMI for 30 min in phosphate-buffered saline pH 7.4 containing 1 mg/mL glucose, 0.4 mM Mg2+, and 1.20 mM Ca2+. Viability was assessed by dimethylthiazol diphenyl tetrazolium bromide (MTT). To distinguish between the two modes of cell death, apoptosis and necrosis, we examined differences in morphological and biochemical changes of cells stained with annexin V- FITC (An) and/or propidium iodide (PI) using two different assays based on flow cytometry Results: The MTT assay revealed the ability of cells to reduce MTT salt to formazan. In the presence of BrI and DMI a significant concentration-dependent decrease of cell viability was observed. The annexin V- FITC binding assay showed a high proportion of apoptotic cells for those treated with BrI (An+/ PI- : 62.3 ± 8.2% vs. 2.1 ± 0.5% of control, P<0.05). The population of PMN treated with DMI produced the highest percentage (An+/IP+: 43.4 ± 5.2 % vs. 0.4 ± 0.3 % of control, P<0.05) of necrotic cells. Apoptotic nuclei were analyzed by PI staining. The cell population in the sub G0/G1 region represents cells with hypodiploidal DNA, an indicator of apoptosis. When cells were incubated with 50 and 100 µM of BrI, the cell population in the sub G0/G1 region increased, suggesting a dose-dependent increase in the population of apoptotic cells. The presence of the pan-inhibitor of caspases (Z-VAD-fmk) showed a significant reduction in cell population in the sub G0/G1 region, indicating less degradation of DNA. Conclusions: Bromide isoespintanol [BrI] induces an apoptotic process in PMN, mediated ­at least in part­ by activation of caspases, although this compound may probably act through other caspase-independent mechanisms as well.

Antecedentes: La inflamación es una respuesta fisiopatológica compleja generada por diferentes estímulos. Recientemente, se han propuesto nuevas estrategias farmacológicas que podrían ser utilizadas para conducir a la resolución de la inflamación mediante el aumento de la apoptosis de células inflamatorias. Objetivo: Estudiar la actividad apoptótica in vitro del isopentanol [ISO] y dos derivados semisintéticos ­bromuro de isoespintanol [BrI] e isoespintanol desmetilado [DMI] ­ en células polimorfonucleares humanas (PMN). Métodos: Las PMN fueron expuestas a diferentes concentraciones de los compuestos durante 30 min en una disolución salina tamponada con fosfato (pH 7,4). La viabilidad celular se evaluó utilizando el ensayo de 3-[4,5-dimetil-tiazol-2-il]-2,5-difenil tetrazolio (MTT). Para distinguir entre los dos modos de muerte celular, la apoptosis y la necrosis, se examinaron las diferencias en los cambios morfológicos y bioquímicos de las células teñidas con anexina V (An) y/o yoduro de propidio (PI) usando dos técnicas de citometría de flujo. Resultados: Mediante el ensayo con MTT, se demostró que los compuestos BrI y DMI disminuyeron significativamente y de manera concentración-dependiente la viabilidad celular. El ensayo de unión con la anexina V-FITC mostró una alta proporción de células apoptóticas en las células tratadas con BrI (An+/PI- : 62,3 ± 8,2% versus 2,1 ± 0,5% del control, P<0,05). El análisis de núcleos apoptóticos se llevó a cabo a través de tinción con PI. La población de células en la región sub G0/G1 representa células con ADN hipodiploidal, que es un indicador de apoptosis. Cuando las células se incubaron con BrI, la población de células en la región sub G0/G1 aumentó, confirmando su mecanismo citotóxico. En presencia de un inhibidor de caspasas (Z-VAD-FMK), se observó una reducción significativa en la población celular en la región sub G0/G1, indicando una menor degradación del ADN. Conclusiones: El bromuro de isoespintanol [BrI], induce un proceso apoptótico en PMN que está mediado ­al menos en parte­ por la activación de las caspasas, aunque este compuesto probablemente podría actuar también a través de otros mecanismos independientes de las caspasas.

Eficacia in vitro de tres endectocidas frente a Triatoma infestans / In vitro efficacy of three endectocides against Triatoma infestans

Introducción: distintos estudios han demostrado el papel preponderante que el peridomicilio cumple en la reinfestación de las viviendas por Triatoma infestans (vinchucas). Con el objetivo de eliminar focos residuales de T. infestans que habitan alrededor de los hogares se han desarrollado distintas estrategias. La administración de diferentes compuestos que tengan actividad contra T. infestans a los animales que habitan zonas cercanas a los domicilios y sirvan como fuente de alimentación a estos insectos, podría ser una buena manera de disminuir el riesgo de reinfestación domiciliaria. Objetivo: evaluar la eficacia in vitro de tres agentes antiparasitarios, doramectina (DRM), ivermectina (IVM) y eprinomectina (EPR) frente a ninfas de quinto estadio de Triatoma infestans. Métodos: se diseñaron alimentadores artificiales en donde se colocó sangre heparinizada y fortificada con distintas concentraciones de los tres endectocidas (100-0,4 ng/mL). Se utilizaron 600 ninfas de quinto estadio de T. infestans durante el experimento. Un grupo de vinchucas fue alimentada con sangre sin tratar (control). Luego de realizada la alimentación se observó el estado de los insectos cada 24 hs. durante el transcurso de una semana. Resultados: los tres endectocidas demostraron actividad frente a ninfas de quinto estadio de T. infestans. Comparando la actividad de las tres moléculas, DRM fue la que exhibió una mayor potencia contra los insectos, inclusive mantuvo su actividad frente T. infestans a 0,4 ng/mL (menor concentración evaluada). En el caso de IVM y EPR comenzaron a perder eficacia a concentraciones por debajo de los 6,25 y 3,15 ng/mL respectivamente, siendo totalmente inactivas a 0,4 ng/mL. Conclusiones: en base a estos resultados podemos aseverar que bajo nuestras condiciones experimentales, tanto IVM, EPR como DRM poseen una alta eficacia in vitro contra T. infestans, siendo la última la más efectiva de las tres evaluadas(AU)

Introduction: various studies have demonstrated the role that areas around the houses play in domiciliary re-infestation by Triatomainfestans (kissing bugs). With the aim of removing residual foci of T. infestans that inhabit in neighboring areas of houses, different strategies have been developed. The administration of different anti-T. infestans compounds to animals living in areas around the houses might be a good way to reduce the risk of domiciliary re-infestation. Objective: to evaluate the in vitro efficacy of three antiparasitic agents, doramectin (DRM), ivermectin (IVM) and eprinomectin (EPR) against fifth instar nymphs of Triatomainfestans. Methods: artificial feeders were designed , which contained heparinized and fortified blood with various concentrations of the three endectocides (100-0.4 ng/mL). We used 600 fifth instar nymphs of T. infestans during the experiment. A group of insects were fed with untreated blood (control). After feeding they were under observation to check their condition every 24 hours for a week. Results: the three molecules showed activity against T. infestans. In comparing the activity of the three molecules, DRM exhibited greater potency against insects, it even kept its activity against T. infestans at 0.4 ng/mL (lowest concentration tested). In the case of EPR and IVM, their efficacy began to lower at concentrations below 6.25 and 3.15 ng/mL respectively, being totally inactive at 0.4 ng/mL concentration. Conclusions: Based on these results, we can assert that under our experimental conditions, IVM, EPR and DRM show in vitro high efficacy against T. infestans, being the latter more effective than the other two molecules(AU)

Anti-inflammatory, anti-oxidant, and apoptotic activities of four plant species used in folk medicine in the Mediterranean basin.

The aim of this research was to study the potential anti-inflammatory activity of myrtle (Myrtus communis), sarsaparilla (Smilax aspera), Arabian or French lavender (Lavandula stoechas), and calamint (Calamintha nepeta) along with their apoptotic effects on the pro-inflammatory cells, and the correlation of these effects with the plants' potential anti-oxidant activity. Myrtle extract exhibited the highest inhibitory activity in the paw oedema induced by carrageenan (60% at 3 h), whereas calamint, lavender, and sarsaparilla produced inhibitions of 49%, 38%, and 47%, respectively. None of them had an effect on the TPA-induced ear oedema. Moreover, all the extracts except sarsaparilla showed different degrees of anti-oxidant activity. Lavender and myrtle at 200 µg/mL decreased cell viability by 63% and 59%, respectively, after 3 h of incubation. Neutrophil elimination through apoptosis could be implicated in the resolution of acute inflammation in the case of lavender, whereas the reduction of reactive oxygen species produced by neutrophils, such as the superoxide anion and the hydroxyl radical, could be implicated in the overall reduction of inflammation. These results may support the traditional use of these plants.

Antioxidant and cytotoxic properties of an aqueous extract from the Argentinean plant Hedeoma multiflorum.

CONTEXT: Hedeoma multiflorum Benth. (Lamiaceae) is widely used in Argentinean popular medicine for digestive and anti-spasmodic purposes. However, knowledge about its pharmacological properties has been poorly investigated. OBJECTIVE: The antioxidant and cytotoxic properties of an aqueous extract from the plant were investigated for the first time. MATERIAL AND METHODS: Scavenging of stable free radicals of 2,2-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS(+)) and 2,2-diphenyl-1-picryl hydrazyl (DPPH), reducing of ferric (III) iron of ferric reducing ability of plasma (FRAP) reagent, and inhibition of lipid peroxidation (LP) of human plasma and rat brain homogenates were assessed. Cytotoxicity was tested on human polymorphonuclear (PMN) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxicity was assessed by flow cytometric techniques. RESULTS: Extract scavenged ABTS(+) and DPPH (1.78 and 0.78 µmol Trolox equivalent/mg dry extract, respectively) and reduced FRAP reagent (0.66 µmol ascorbic acid equivalent/mg dry extract). LP of human plasma and rat brain was also inhibited in a dose-dependent way (inhibitory concentration 50%=27.0 and 86.0 µg/mL, respectively). Extract is rich in polyphenol compounds (0.96 ± 0.08 µmol equivalent caffeic acid/mg dry matter). Treatment of PMN decreased significantly the cell ability to reduce the MTT salt and increased the hypodiploid nuclei from 4 to 18% quantified using propidium iodide (PI). In the annexin V-Fluorescein isothiocyanate (annexin V-FITC) assay, 26% of treated cells were annexin V-FITC positive and PI negative. Using the 3,3'-dihexyloxacarbocyanine iodide uptake method, the negative fraction of cells was calculated as 29%. DISCUSSION AND CONCLUSION: H. multiflorum extract was found to have a significant antioxidant and pro-apoptotic activities, and a great potential as a source of healthy products.

Anti-inflammatory and apoptotic activities of pomolic acid isolated from Cecropia pachystachya.

The dichloromethane extract and pomolic acid ( 5) obtained from leaves of Cecropia pachystachya both reduced carrageenan-induced paw oedema in mice. Interestingly, while the triterpenoid inhibited the in vivo production of interleukin-1beta by 39 %, it had no effect on tumour necrosis factor-alpha production. We also demonstrated that both the dichloromethane extract and 5 inhibited the viability of human polymorphonuclear (PMN) cells in a time- and dose-dependent fashion. The PMN membrane integrity was determined with the aid of flow cytometry by means of the exclusion of propidium iodide as assay. Although the cell membrane integrity was altered, neither the extract nor 5 produced cellular necrosis. Moreover, the development of hypodiploid nuclei and DNA fragmentation in the PMN cells were both dependent on dose and time. Finally, in the annexin V-FITC binding assay, compound 5 increased the total of apoptotic cells by 42 % at 100 microM and by 71 % at 200 microM with respect to the control group. In conclusion, both the dichloromethane extract of ambay and isolated compound 5 inhibit the viability of PMN cells through apoptosis. Since they can regulate human neutrophil functions in this way, it is likely that these substances can also limit inflammation.