Further insecticidal activities of essential oils from Lippia sidoides and Croton species against Aedes aegypti L.

This study assessed new insecticidal activities of essential oils from Lippia sidoides and Croton species (Croton zehntneri, Croton nepetaefolius, Croton argyrophylloides, and Croton sonderianus) against Aedes aegypti mosquito. In addition, the acute toxicity upon mice was determined. All essential oils showed inhibition of egg hatching, with IC₅₀ values ranging from 66.4 to 143.2 μg?mL^?1, larvicidal activity with LC₅₀ ranging from 25.5 to 94.6 μg?mL^?1, and pupicidal action with PC₅₀ ranging from 276.8 to over 500 μg?mL^?1. Only L. sidoides, C. zehntneri, and C. argyrophylloides essential oils were able to inhibit the oviposition of female gravid mosquitoes with OD₅₀ values of 35.3, 45.3, and 45.8 μg?mL^?1, respectively. Oral acute toxicity in mice showed that C. sonderianus and C. argyrophylloides oils are nontoxic (LD₅₀?>?6,000 mg.kg^?1) while C. nepetaefolius, C. zehntneri, and L. sidoides oils are moderately toxic (LD₅₀ 3,840; 3,464, and 2,624 mg.kg^?1, respectively). The results indicate that these oils are promising sources of bioactive compounds, showing low or no toxicity to mammals.     

Evaluation of time toxicity, residual effect, and growth-inhibiting property of Carapa guianensis and Copaifera sp. in Aedes aegypti

Oils of Carapa guianensis and Copaifera spp. are well-known in the Amazonian region as natural insect repellents, and studies have reported their efficiency as larvicide against some mosquito species. However, toxicity persistence and effect on mosquito development have not yet been evaluated. Thus, the objective of this study was to evaluate the initial time of larvicidal activity, residual effect, and the effect of very low concentrations of these oils on Aedes aegypti. Different concentrations of the oils were used to evaluate the initial time of larval mortality and residual effect, as well as, the development of larvae, pupae, and adults. Results demonstrated that the lethal effect started mainly between the first 2 and 3 h of larvae exposure to oils, when using concentrations which ranged from 500 mg/L of C. guianensis and 90 mg/L of Copaifera sp. The toxic effect remained with total efficiency (100% mortality) until the sixth day for Copaifera sp. and 12th day for C. guianensis. When using sublethal dosages (ranging from 140 mg/L of C. guianensis to 26 mg/L of Copaifera sp.) mortality was observed after the larval molt. Also, imperfection of pupae and adult development and unsuccessful emergence of adults were observed. A product of botanical origin that could break the development of immature stage of mosquitoes and inhibit the emergence of adults should be essential in vector control. Thus, our results provide new information for a better understanding in using C. guianensis and Copaifera sp. oils with a potential to be used as a natural insecticide.     

Citrus essential oils and four enantiomeric pinenes against Culex pipiens (Diptera: Culicidae)

The aim of this study was to evaluate the toxicity of pinenes (entantiomers of α- and β-) and essential oils from Greek plants of the Rutaceae family against the mosquito larvae of Culex pipiens (Diptera: Culicidae). Essential oils were isolated by hydrodistillation from fruit peel of orange (Citrus sinensis L.), lemon (Citrus limon L.), and bitter orange (Citrus aurantium L.). The chemical composition was determined by gas chromatography/mass spectrometry (GC/MS) analysis. Citrus essential oils contained in high proportion limonene and in lower quantities p-menthane molecules and pinenes. The insecticidal action of these essential oils and entantiomers of their pinenes on mosquito larvae was evaluated. Plant essential oils exhibited strong toxicity against larvae with the LC₅₀ values ranging from 30.1 (lemon) to 51.5 mg/L (orange) depending on Citrus species and their composition. Finally, the LC₅₀ value of pinenes ranging from 36.53 to 66.52 mg/L indicated an enantioselective toxicity only for the β-pinene entantiomer.     

The use of essential oils in histologic practice

Summary Clearing of microscopic sections as well as extraction of celloidin from them can be successfully accomplished with the use of such essential oils asCymbopogon citratus,Ocimum gratissimum,Eucalyptus citriodare, andPelargonium roseum instead of the usual oil of cloves. For differentiating and staining nerve cell preparations, good results have been obtained with the use of the essential oils ofMelaleuca alternifolia andPogostemon patchouli instead of cajuput terpene oil. Presented by Active Member Acad. Med. Sci. USSR N. G. Khiopin     

Antileishmanial activity and ultrastructural alterations of Leishmania (L.) chagasi treated with the calcium channel blocker nimodipine

In a search for novel antileishmanial drugs, we investigated the activity of the calcium channel blocker nimodipine against Leishmania spp. and explored the ultrastructural damages of parasites induced by nimodipine after a short period of incubation. Nimodipine was highly effective against promastigotes and intracellular amastigotes of Leishmania (L.) chagasi, with 50% inhibitory concentration values of 81.2 and 21.5 μM, respectively. Nimodipine was about fourfold more effective than the standard pentavalent antimony against amastigotes and showed a Selectivity Index of 4.4 considering its mammalian cells toxicity. Leishmania (L.) amazonensis and Leishmania (L.) major promastigotes were also susceptible to nimodipine in a range concentration between 31 and 128 μM. Ultrastructural studies of L. (L.) chagasi revealed intense mitochondria damage and plasma membrane blebbing, resulting in a leishmanicidal effect as demonstrated by the lack of mitochondrial oxidative metabolism. The amastigote-killing effect suggests other mechanism than macrophage activation, as no upregulation of nitric oxide was seen. This calcium channel blocker is an effective in vitro antileishmanial compound and if adequately studied could be used as a novel drug candidate or as a novel drug lead compound for drug design studies against leishmaniasis.     

In vitro activity of the essential oil of <Emphasis Type="Italic">Cymbopogon citratus</Emphasis> and its major component (citral) on <Emphasis Type="Italic">Leishmania amazonensis</Emphasis>

Leishmaniasis causes considerable mortality throughout the world, affecting more than 12 million people. Cymbopogon citratus (DC) Stapf, Family Poaceae, is a widely used herb in tropical countries and is also known as a source of ethnomedicines. In this study, the inhibitory effect and the morphological and ultrastructural alterations on Leishmania amazonensis by the essential oil (EO) of C. citratus and its main constituent, citral, were evaluated. The results showed that the antiproliferative activity of EO on promastigotes and axenic amastigotes, and intracellular amastigote forms of L. amazonensis was significantly better than citral, and indicated a dose-dependent effect. Neither compound showed a cytotoxic effect on macrophage strain J774G8. The promastigote forms of L. amazonensis underwent remarkable morphological and ultrastructural alterations compared with untreated cultures. These alterations were visible by light, scanning, and transmission electron microscopy of promastigotes treated with EO and citral at concentrations corresponding to the IC₅₀ (1.7 and 8.0 μg/ml) and IC₉₀ (3.2 and 25 μg/ml), respectively, after 72 h of incubation. This study revealed that citral-rich essential oil from C. citratus has promising antileishmanial properties, and is a good candidate for further research to develop a new anti-protozoan drug.     

Chemical composition and toxicity of the essential oils from Cunila species (Lamiaceae) on the cattle tick Rhipicephalus (Boophilus) microplus

The essential oils obtained from the aerial parts of five species of Cunila (Lamiaceae) native to Southern Brazil were analyzed by GC and GC/MS. The oil of Cunila angustifolia was characterized by sabinene; Cunila incana is rich in α-pinene and β-pinene, Cunila spicata and Cunila microcephala presented menthofuran as the main component, and in the essential oil of Cunila incisa, the major component was 1,8-cineole. Laboratory tests were carried out to determine the effect of the essential oils of the above cited plants on larvae of the cattle tick Riphicephalus (Boophilus) microplus. C. angustifolia, C. incana, and C. spicata were the most active samples killing almost the totality of the larvae. C. incisa and C. microcephala showed low acaricidal effect.     

Oviposition deterrent and ovicidal activities of seven herbal essential oils against female adults of housefly,Musca domestica L.

The oviposition deterrent and ovicidal of seven herbal essential oils derived from Citrus sinensis, Cymbopogon citratus, Eucalyptus glubulus, Illicium verum, Lavandula angustifolia, Mentha piperita, and Zingiber cussumunar were assessed against the gravid female of housefly, Musca domestica L., under laboratory conditions and compared with commercial insecticide (10 % w/v cypermethrin). They were assayed at three concentrations (1.0, 5.0, and 10.0 %) where plastic cups containing 1 ml of desired oil concentration and cotton pad soaked with 10 ml of milk solution (10 % w/v) were used as oviposition substrate. The 0.1 ml of deferent concentrations was dropped on ten housefly eggs, which were used for ovicidal activity. The number of eggs laid and the hatched larvae in each cup was recorded to evaluate the oviposition deterrent and ovicidal activities of the herbal essential oils. High concentration (10 %) of herbal essential oils showed high percent effective repellency (ER). The 10 % I. verum oil caused complete oviposition deterrence (100 % ER, oviposition activity index (OAI)?=??1.0), followed by Z. cussumunar, M. piperita, L. angustifolia, C. citratus, C. sinensis, and E. glubulus oils with 97.20, 88.55, 88.14, 87.93, 76.68, and 57.00 % ER, respectively. As the concentration of herbal essential oils increased from 1.0, 5.0, and up to 10.0 % concentration, the hatching rate decreased. Ten percent I. verum oil gave the maximum inhibiting rate at 97.3 % (LC₅₀ value of 6.85 %); in addition, the other herbal essential oils showed the minimum inhibiting rate of 3.3–22.7 %. On the other hand, cypermethrin 10 % w/v showed complete oviposition deterrence (100 % ER, OAI?=??1.0) and ovicidal activity (100 % inhibiting rate). Our data showed that I. verum oil have high potential of oviposition deterrence and ovicide housefly control.     

Acaricidal activity and chemical composition of the essential oil from three Piper species

The chemical composition of the essential oil obtained by hydrodistillation of the aerial parts of Piper amalago, Piper mikanianum, and Piper xylosteoides was elucidated by gas chromatography (GC) and GC/mass spectrometry analyses. P. mikanianum and P. xylosteoides essential oils presented phenylpropanoids as their main compounds (67.89% and 48.53%, respectively) whereas P. amalago was rich in monoterpene and sesquiterpene hydrocarbons (84.95%). The essential oils obtained were investigated for their effect on newly hatched larvae of the cattle tick, Rhipicephalus (Boophilus) microplus. The essential oil of P. mikanianum (LC₅₀ 2.33 μL/mL) was more active than that of P. xylosteoides (LC₅₀ 6.15 μL/mL) against the larvae, while the oil of P. amalago was inactive. These results suggest that phenylpropanoids, mainly apiol and safrole, are responsible for the acaricidal activity.     

Biological activity of selected Lamiaceae and Zingiberaceae plant essential oils against the dengue vector Aedes aegypti L. (Diptera: Culicidae)

The larvicidal activity of hydrodistillate extracts from Mentha piperita L. Ocimum basilicum L. Curcuma longa L. and Zingiber officinale L. were investigated against the dengue vector Aedes aegypti L. (Diptera: Culicidae).The results indicated that the mortality rates at 80, 100, 200 and 400 ppm of M. piperita, Z. officinale, C. longa and O. basilicum concentrations were highest amongst all concentrations of the crude extracts tested against all the larval instars and pupae of A. aegypti. Result of log probit analysis (at 95% confidence level) revealed that lethal concentration LC₅₀ and LC₉₀ values were 47.54 and 86.54 ppm for M. piperita, 40.5 and 85.53 ppm for Z. officinale, 115.6 and 193.3 ppm for C. longa and 148.5 and 325.7 ppm for O. basilicum, respectively. All of the tested oils proved to have strong larvicidal activity (doses from 5 to 350 ppm) against A. aegypti fourth instars, with the most potent oil being M. piperita extract, followed by Z. officinale, C. longa and O. basilicum. In general, early instars were more susceptible than the late instars and pupae. The results achieved suggest that, in addition to their medicinal activities, Lamiaceae and Zingiberaceae plant extracts may also serve as a natural larvicidal agent.     

Repellence of essential oils of aromatic plants growing in Argentina against Aedes aegypti (Diptera: Culicidae)

Mosquitoes are vectors of pathogens to humans and domestic animals and may also have economical impacts. One approach to prevent mosquito-borne diseases is bite deterrence through the application of repellents. Currently, there is an interest to search for alternative bioactive products to the synthetic active ingredients most widely used in insect repellents. Repellence against Aedes aegypti of essential oils extracted from Acantholippia salsoloides, Aloysia catamarcensis, Aloysia polystachya, Lippia integrifolia, Lippia junelliana (Verbenaceae), Baccharis salicifolia, Euphatorium buniifolium, and Tagetes filifolia (Asteraceae) were assessed. Tests were conducted by alternatively exposing untreated and treated forehand to the mosquitoes and counting probing attempts. All essential oils tested were significantly repellent against A. aegypti when compared to untreated controls; L. junelliana was the most repellent and T. filifolia was the least based on the response of the mosquitoes to different concentrations of the essential oils (EO). Repellence may be attributed to the respective main components of each EO.     

Anti-Giardia activity of phenolic-rich essential oils: effects of Thymbra capitata, Origanum virens, Thymus zygis subsp. sylvestris, and Lippia graveolens on trophozoites growth, viability, adherence, and ultrastructure

The present work evaluates the anti-Giardia activity of phenolic-rich essential oils obtained from Thymbra capitata, Origanum virens, Thymus zygis subsp. sylvestris chemotype thymol, and Lippia graveolens aromatic plants. The effects were evaluated on parasite growth, cell viability adherence, and morphology. The tested essential oils inhibited the growth of Giardia lamblia. T. capitata essential oil is the most active followed by O. virens, T. zygis subsp. sylvestris, and L. graveolens oils. The tested essential oils at IC50 (71–257) μg/ml inhibited parasite adherence (p < 0.001) since the first hour of incubation and were able to kill almost 50% of the parasites population in a time-dependent manner. The main ultrastructural alterations promoted by essential oils were deformations in typical trophozoite appearance, often roundly shape, irregular dorsal and ventral surface, presence of membrane blebs, electrodense precipitates in cytoplasm and nuclei, and internalization of flagella and ventral disc. Our data suggest that essential oils induced cell death probably by processes associated to the loss of osmoregulation caused by plasmatic membrane alterations. Experiments revealed that the essential oils did not present cytotoxic effects in mammalian cells. In conclusion, T. capitata, O. virens, T. zygis subsp. sylvestris chemotype thymol, and L. graveolens essential oils have antigiardial activity in vitro and seem to have potential for the treatment of the parasitic disease caused by the protozoan G. lamblia.     

Antistaphylococcal activity of Inula helenium L. root essential oil: eudesmane sesquiterpene lactones induce cell membrane damage

The purpose of this study was to investigate the inhibitory/bactericidal activity and cell membrane effects of the hydrodistilled essential oil of Inula helenium L. roots against Staphylococcus aureus. Additionally, detailed chemical investigation was done in order to pinpoint the most active oil constituents and also the parts of these molecules responsible for their antimicrobial effect. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the broth microdilution method. The membrane-active nature of this oil was investigated by measuring the culture turbidity, leakage of phosphates, and 260-nm-absorbing material, together with lysis of the exposed cells. Finally, the effect of the oil on the cells was visualized using scanning electron microscopy (SEM). The chemical composition of the essential oil was analyzed using gas chromatography-mass spectrometry (GC-MS) and preparative medium-pressure liquid chromatography (MPLC). Chemical modification of the oil was performed using catalytic hydrogenation (H₂, Pd/C) and reduction with NaBH₄. The MIC and MBC values were 0.01 μl mL⁻¹ and 0.02 μl mL⁻¹, respectively. Membrane damage was demonstrated through increased permeability (phosphates and nucleic acid leakage), followed by lysis of the exposed cells, captured on SEM images. The most active constituents were alantolactone, isoalantolactone, and diplophyllin. The essential oil showed very potent antistaphylococcal activity, with obvious membrane-damaging effects. Sesquiterpene lactones were found to be the most active principles of the oil, whose eudesmane core olefinic bonds, along with the α,β-methylene-lactone ring, are essential structural parts responsible for the exhibited antimicrobial activity.     

Larvicidal potential of medicinal plant extracts against Anopheles subpictus Grassi and Culex tritaeniorhynchus Giles (Diptera: Culicidae)

Mosquito control is facing a threat due to the emergence of resistance to synthetic insecticides. Insecticides of botanical origin may serve as suitable alternative biocontrol techniques in the future. The acetone, chloroform, ethyl acetate, hexane, methanol and petroleum ether extracts of leaf, flower and seed of Cassia auriculata L., Leucas aspera (Willd.), Rhinacanthus nasutus KURZ., Solanum torvum Swartz and Vitex negundo Linn. were tested against fourth instar larvae of malaria vector, Anopheles subpictus Grassi and Japanese encephalitis vector, Culex tritaeniorhynchus Giles (Diptera: Culicidae). The larval mortality was observed after 24 h of exposure. All extracts showed moderate larvicidal effects; however, the highest mortality was found in leaf petroleum ether, flower methanol extracts of C. auriculata, flower methanol extracts of L. aspera and R. nasutus, leaf and seed methanol extracts of S. torvum and leaf hexane extract of V. negundo against the larvae of A. subpictus (LC₅₀ = 44.21, 44.69, 53.16, 41.07, 35.32, 28.90 and 44.40 ppm; LC₉₀ = 187.31, 188.29, 233.18, 142.66, 151.60, 121.05 and 192.11 ppm, respectively) and against the larvae of C. tritaeniorhynchus (LC₅₀ = 69.83, 51.29, 81.24, 71.79, 44.42, 84.47 and 65.35 ppm; LC₉₀ = 335.26, 245.63, 300.45, 361.83, 185.09, 351.41 and 302.42 ppm, respectively). These results suggest that the leaf petroleum ether, flower methanol extracts of C. auriculata, leaf and seed methanol extracts of S. torvum and leaf hexane extract of V. negundo have the potential to be used as an ideal eco-friendly approach for the control of the A. subpictus and C. tritaeniorhynchus. This is the first report on the mosquito larvicidal activity of the medicinal plant extracts.     

Furazolidone is a selective in vitro candidate against Leishmania (L.) chagasi: an ultrastructural study

The current treatment for leishmaniasis is unsatisfactory due to toxic side effects, high cost, and problems with drug resistance. Various approaches have been used to identify novel drug candidates to treat Leishmania sp. parasites including the use of re-purposed drugs. Furazolidone is a nitrofuran derivative with antiprotozoal and antibacterial activity and is used for the treatment of giardiasis. In the present work, we determined the in vitro antileishmanial activity of furazolidone and its ability to induce ultrastructural alterations of parasites. Promastigotes of Leishmania (L.) chagasi, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) amazonensis were highly susceptible to furazolidone, with IC₅₀ values ranging between 0.47 and 0.73 μg/mL. Furazolidone was also very effective against L. chagasi intracellular amastigotes, and despite mammalian cytotoxicity, the selectivity index was 8.0 in human monocytes. The drug also had limited toxicity in mice erythrocytes. Furazolidone demonstrated specific activity against Leishmania, a potential consequence of the lack of macrophage nitric oxide activation. As determined by electron transmission microscopy, drug treatment induced severe damage to the parasite mitochondria and nucleus. This older oral drug is an effective agent for the treatment of L. (L.) chagasi in vitro and is a novel candidate for further experimental studies.     

Chemical composition and larvicidal evaluation of Mentha, Salvia, and Melissa essential oils against the West Nile virus mosquito Culex pipiens

The volatile metabolites of wild-growing Mentha spicata, M. longifolia, M. suaveolens, Melissa officinalis, Salvia fruticosa, S. pomifera subsp. calycina, and S. pomifera subsp. pomifera from Greece were determined by gas chromatography and gas chromatography–mass spectrometry. The insecticidal properties of the analyzed essential oils were screened on Culex pipiens larvae. Additionally two of the main components of the essential oils, piperitenone oxide and 1,8-cineole were assayed against C. pipiens in order to define the affiliation between them and the larvicidal properties of the oils. The most effective oils were M. suaveolens (major constituent piperitenone oxide, 62.4%), M. spicata (piperitenone oxide, 35.7% and 1,8-cineole, 14.5%) and M. longifolia—Central Greece (piperitenone oxide, 33.4%; 1,8-cineole, 24.5% and trans-piperitone epoxide, 17.4%), which exhibited LC₅₀ values ranging from 47.88 to 59.33 mg l⁻¹. Medium activity revealed the oils of M. officinalis (terpin-4-ol, 15.8%; caryophyllene oxide, 13.2%; sabinene, 12.9%; β-pinene, 12.1%; and trans-caryophyllene, 10.2%), M. longifolia—Southern Greece (carvone, 54.7% and limonene 20.0%), S. pomifera subsp. pomifera (trans-caryophyllene, 22.5% and trans-thujone, 21.0%), S. pomifera subsp. calycina—West Southern Greece (trans-thujone, 56.1% and 1,8-cineole, 10.4%), and S. fruticosa—population 2 (camphor, 23.1%; α-pinene, 12.7%; and borneol, 12.6%), with LC₅₀ values ranging from 78.28 to 91.45 mg l⁻¹. S. pomifera subsp. calycina (Central Greece) essential oil (trans-thujone, 26.5% and cis-thujone, 12.0%) presented rather low activity (LC₅₀ values 140.42 mg l⁻¹), while S. fruticosa—population 1 (1,8-cineole, 31.4% and camphor, 22.6%) was the only inactive oil. Additionally, the constituent piperitenone oxide was found to be highly active (LC₅₀ values 9.95 mg l⁻¹), whereas 1,8-cineole revealed no toxicity.     

Eucalyptus essential oil toxicity against permethrin-resistant Pediculus humanus capitis (Phthiraptera: Pediculidae)

During the past decades, chemical control against the head louse Pediculus humanus capitis De Geer has been based in the application of products containing permethrin. The repetitive overuse of pediculicides has resulted in the development of high levels of resistance to one or more of these products worldwide. Essential oils obtained from aromatic plants like Eucalyptus are good and safe alternatives due to their low toxicity to mammals and easy biodegradability. In the present study, we reported the chemical composition of Eucalyptus dunnii and Eucalyptus gunni, and the fumigant activity of five Eucalyptus essential oils and their main compounds against permethrin-resistant head lice from Argentina. The most effective essential oils were Eucalyptus sideroxylon, Eucalyptus globulus ssp globulus, and Eucalyptus globulus ssp maidenii, with knockdown time 50% (KT₅₀) values of 24.75, 27.73, and 31.39 min. A linear regression analysis between percentage of 1,8-Cineole and KT₅₀ values of the essential oils showed a significant correlation at a p < 0.01. Since Eucalyptus essential oils showed to be effective against head lice and are classified as safer compounds, they can be employed into pediculicide formulations.     

Fast high yield of pure Leishmania (Leishmania) infantum axenic amastigotes and their infectivity to mouse macrophages

Leishmania (L.) infantum (syn. Leishmania chagasi) is a dimorphic protozoan parasite that lives in promastigote and amastigote form in its sandfly vector and mammalian hosts, respectively. Here, we describe an in vitro culture system for the generation of a pure population of L. infantum axenic amastigotes after only 4 days incubation in culture medium supplemented with fetal calf serum, human urine, l-glutamine, and HEPES at 37oC (pH 5.5). Ultrastrutural analysis and infection assays in two macrophage populations (Kupffer cells (KUP) and peritoneal macrophages (PM)) infected with axenic amastigotes demonstrated that they maintained morphological and biochemical (A2 expression) features and a similar infection pattern to tissue-derived L. infantum amastigotes. The susceptibility of the macrophage lines to axenic or tissue-derived amastigotes and promastigotes was investigated. We found a completely different susceptibility profile for KUP and PM. Liver macrophages, both KUP and immigrant macrophages, are intimately involved in the response to L. infantum infection; this difference in susceptibility is probably related to their capacity to eliminate these parasites. Our in vitro system was thus able to generate axenic amastigotes that resemble tissue-derived amastigotes both in morphology and infectivity pattern; this will help in further investigation of the biological characteristics of the host–parasite relationship as well as the process of pathogenesis.     

Serine protease activities in Leishmania (Leishmania) chagasi promastigotes

The present work reports the isolation, biochemical characterization, and subcellular location of serine proteases from aqueous, detergent soluble, and culture supernatant of Leishmania chagasi promastigote extracts, respectively, LCSII, LCSI, and LCSIII. The active enzyme molecular masses of LCSII were about 105, 66, and 60 kDa; of LCSI, 60 and 58 kDa; and of LCSIII, approximately 76 and 68 kDa. Optimal pH for the enzymes was 7.0 for LCSI and LCSIII and 8.5 for LCSII, and the optimal temperature for all enzymes was 37°C, using α-N-ρ-tosyl-l-arginine methyl ester as substrate. Assay of thermal stability indicated that LCSIII is the more stable enzyme. Hemoglobin, bovine serum albumin, and ovalbumin were hydrolyzed by LCSII and LCSI but not by LCSIII. Inhibition studies suggested that enzymes belong to the serine protease class modulated by divalent cations. Rabbit antiserum against 56-kDa serine protease of Leishmania amazonensis identified proteins in all extracts of L. chagasi. Furthermore, immunocytochemistry demonstrated that serine proteases are located in flagellar pocket region and cytoplasmic vesicles of L. chagasi promastigotes. These findings indicate that L. chagasi serine proteases differ from L. amazonensis proteases and all known flagellate proteases, but display some similarities with serine proteases from other Leishmania species, suggesting a conservation of this enzymatic activity in the genus.     

Occurrence of antibodies anti-Neospora caninum, anti-Toxoplasma gondii, and anti-Leishmania chagasi in serum of dogs from Pará State, Amazon, Brazil

A cross-sectional study was conducted to determine the occurrence of anti-Toxoplasma gondii, anti-Neospora caninum, and anti- Leishmania chagasi antibodies in dogs of the state of Pará, Brazil. For this purpose, 129 blood samples were collected from dogs of different ages and gender. Samples of 72 dogs were collected from 39 rural properties from 19 municipalities, and 57 samples were from stray dogs, collected after captivity by the Center of Zoonosis Control from the municipality of Santarém. The sera were analyzed for anti-T. gondii and anti-N. caninum antibodies by indirect fluorescent antibody tests with cutoff values of 1:16 and 1:50, respectively. For the presence of L. chagasi antibodies, enzyme-linked immunosorbent assay was used and positive results were confirmed by immunochromatographic method using the recombinant antigen K39. Of the total of 129 dogs, 90 (69.8%) were positive for T. gondii, 16 (12.4%) for N. caninum, and 30 (23.3%) for L. chagasi. Antibodies for all three parasites were found simultaneously in seven dogs (5.4%), mostly in urban dogs (six of seven). No association was observed related to gender and location (urban or rural) of dogs and occurrence of N. caninum and T. gondii antibodies although, regarding L. chagasi, higher prevalence was found in females (P < 0.02) and in dogs from urban location (P < 0.001). From the 39 farms, in 30 (76.9%) at least one dog was positive for T. gondii or N. caninum or both. Higher occurrence of Leishmania antibodies was observed in N. caninum-negative dogs (P < 0.05).