Blood parasites (Kinetoplastida: Trypanosomatidae) infecting birds in the Brazilian Amazon / Parásitos sanguíneos (Kinetoplastida: Trypanosomatidae) que infectan aves en la Amazonía brasileña

Abstract Introduction: Trypanosomes are hemoparasites that can be observed circulating in the peripheral blood of birds. Parasitological studies in birds in their natural environment are neglected, but are important for research relating to transmission, maintenance of the biological cycle, and abundance, among other parasitological aspects. Objective: To describe infections by Trypanosoma sp. in birds in the Brazilian Amazon, as well as the prevalence, morphological and morphometric characteristics of this hemoparasite. Methods: In the Tapajós National Forest, we captured a total of 125 birds, mostly from the order Passeriformes. We obtained blood samples from the ulnar vein using sterile insulin needles, and aliquots of blood using a microhematocrit capillary tube. We made blood smears in triplicate and stained with the Giemsa method. We viewd the morphotypes of the Trypanosoma sp. under the light microscope with objective lenses of 40 X and 100 X. To determine the morphometric characteristics of Trypanosomatidae, we used the Zen Blue Edition 2 software package. Results: We observed the presence of hemoparasites in the trypomastigote form in specimens of Thamnophilidae, Dendrocolaptidae and Conopophagidae, with low prevalence. Only one morphotype of Trypanosoma sp. was detected and measurement. Conclusions: We report the infection by Trypanosoma sp. in species of ecological importance, such as Phlegopsis nigromaculata, endangered in Brazil. The morphology and morphometry of the morphotype found could contribute to more detailed descriptions of these hemoparasites.

Resumen Introducción: Los tripanosomas son hemoparásitos que pueden observarse circulando en la sangre periférica de las aves. Los estudios parasitológicos en aves en el medio natural son escasos, pero son importantes para la investigación relacionada con la transmisión, el mantenimiento del ciclo biológico y la abundancia, entre otros aspectos parasitológicos. Objetivo: Describir infecciones por Trypanosoma sp. en aves de la Amazonia brasileña, así como la prevalencia, características morfológicas y morfométricas de este hemoparásito. Métodos: En la Floresta Nacional de Tapajós, capturamos un total de 125 aves, la mayoría del orden Passeriformes. Obtuvimos muestras de sangre por punción de la vena cubital del ala con agujas estériles de insulina. Con un tubo capilar microhematocrito, obtuvimos alícuotas de sangre. Realizamos frotis de sangre por triplicado y teñimos con el método de Giemsa. Visualizamos los morfotipos de Trypanosoma sp. al microscopio óptico con lentes objetivos de 40 X y 100 X. Para determinar las características morfométricas de Trypanosomatidae, usamos el paquete informático Zen Blue Edition 2. Resultados: Observamos la presencia de hemoparásitos en la forma tripomastigote en ejemplares de la familia de aves Thamnophilidae, Dendrocolaptidae y Conopophagidae, con baja prevalencia. Solo detectamos un morfotipo de Trypanosoma sp. Conclusión: Reportamos la infección por Trypanosoma sp. en especies de importancia ecológica, como Phlegopsis nigromaculata en peligro de extinción en Brasil. La morfología y morfometría del morfotipo encontrado puede contribuir con descripciones más detalladas de estos hemoparásitos.

Activity of Piperaceae extracts and fractions in the control of Phytomonas serpens / Atividade de extratos e frações de Piperaceae no controle de Phytomonas serpens

Protozoa of the genus Phytomonas are harmful parasites to several agricultural crops of economic importance. Due to their recognized biological activity, crude extracts of Piper aduncum, P. crassinervium, P. hispidum, and P. amalago leaves, were tested using the microdilution plate technique to assess the antiparasitic potential against Phytomonas serpens. Results showed that the ethanolic crude extract of P. crassinervium and P. amalago presented the best inhibitory concentration for 50% of the cells (IC50), 16.5 µg mL-1 in chloroform phase, and 18 µg mL-1 in aqueous phase, respectively, after 48 h treatment. Cytotoxicity analyses were performed using the colorimetric method of sulforhodamine-B in LLCMK2 mammalian cells. The chloroform phase of P. crassinervium was subjected to the fractionation process, in which the ethyl acetate and dichloromethane fractions obtained better IC50 values. Scanning electron microscopy (SEM) images showed alterations in the cell membrane of the treated parasites. The data obtained indicate a potential antiparasitic effect of the Piper species analyzed against P. serpens, being considered promising candidates for formulations of bioproducts to control the parasite.(AU)

Protozoários do gênero Phytomonas são parasitas prejudiciais a várias culturas agrícolas de importância econômica. Devido a sua atividade biológica reconhecida, extratos brutos de folhas de Piper aduncum, P. crassinervium, P. hispidum e P. amalago, foram testadas pela técnica de microdiluição em placa para avaliar o seu potencial antiparasitário contra Phytomonas serpens. Os resultados mostraram que o extrato bruto etanólico de P. crassinervium e P. amalago apresentaram as melhores concentrações inibitórias para 50% das células (IC50), 16,5 µg mL-1 na fase clorofórmio e 18 µg mL-1 na fase aquosa, respectivamente, após 48 h de tratamento. Análises de citotoxicidade foram realizadas através do método colorimétrico da sulforodamina-B, em células de mamíferos LLCMK2. A fase clorofórmio de P. crassinervium foi submetida ao processo de fracionamento, no qual as frações acetato de etila e diclorometano obtiveram melhores valores de IC50. Imagens de microscopia eletrônica de varredura (MEV) mostraram alterações na membrana celular dos parasitas tratados com fase aquosa de P. amalago. Os dados obtidos indicam potencial efeito antiparasitário das espécies de Piper analisadas contra P. serpens, sendo consideradas candidatas promissoras para formulações de bioprodutos para controle do parasito.(AU)

Natural infection by the protozoan Leptomonas wallacei impacts the morphology, physiology, reproduction, and lifespan of the insect Oncopeltus fasciatus.

Trypanosomatids are protozoan parasites that infect thousands of globally dispersed hosts, potentially affecting their physiology. Several species of trypanosomatids are commonly found in phytophagous insects. Leptomonas wallacei is a gut-restricted insect trypanosomatid only retrieved from Oncopeltus fasciatus. The insects get infected by coprophagy and transovum transmission of L. wallacei cysts. The main goal of the present study was to investigate the effects of a natural infection by L. wallacei on the hemipteran insect O. fasciatus, by comparing infected and uninfected individuals in a controlled environment. The L. wallacei-infected individuals showed reduced lifespan and morphological alterations. Also, we demonstrated a higher infection burden in females than in males. The infection caused by L. wallacei reduced host reproductive fitness by negatively impacting egg load, oviposition, and eclosion, and promoting an increase in egg reabsorption. Moreover, we associated the egg reabsorption observed in infected females, with a decrease in the intersex gene expression. Finally, we suggest alterations in population dynamics induced by L. wallacei infection using a mathematical model. Collectively, our findings demonstrated that L. wallacei infection negatively affected the physiology of O. fasciatus, which suggests that L. wallacei potentially has a vast ecological impact on host population growth.

Impact of nutritional stress on the honeybee colony health.

Honeybees Apis mellifera are important pollinators of wild plants and commercial crops. For more than a decade, high percentages of honeybee colony losses have been reported worldwide. Nutritional stress due to habitat depletion, infection by different pests and pathogens and pesticide exposure has been proposed as the major causes. In this study we analyzed how nutritional stress affects colony strength and health. Two groups of colonies were set in a Eucalyptus grandis plantation at the beginning of the flowering period (autumn), replicating a natural scenario with a nutritionally poor food source. While both groups of colonies had access to the pollen available in this plantation, one was supplemented with a polyfloral pollen patty during the entire flowering period. In the short-term, colonies under nutritional stress (which consumed mainly E. grandis pollen) showed higher infection level with Nosema spp. and lower brood and adult bee population, compared to supplemented colonies. On the other hand, these supplemented colonies showed higher infection level with RNA viruses although infection levels were low compared to countries were viral infections have negative impacts. Nutritional stress also had long-term colony effects, because bee population did not recover in spring, as in supplemented colonies did. In conclusion, nutritional stress and Nosema spp. infection had a severe impact on colony strength with consequences in both short and long-term.

Cell death pathways in pathogenic trypanosomatids: lessons of (over)kill.

Especially in tropical and developing countries, the clinically relevant protozoa Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (sleeping sickness) and Leishmania species (leishmaniasis) stand out and infect millions of people worldwide leading to critical social-economic implications. Low-income populations are mainly affected by these three illnesses that are neglected by the pharmaceutical industry. Current anti-trypanosomatid drugs present variable efficacy with remarkable side effects that almost lead to treatment discontinuation, justifying a continuous search for alternative compounds that interfere with essential and specific parasite pathways. In this scenario, the triggering of trypanosomatid cell death machinery emerges as a promising approach, although the exact mechanisms involved in unicellular eukaryotes are still unclear as well as the controversial biological importance of programmed cell death (PCD). In this review, the mechanisms of autophagy, apoptosis-like cell death and necrosis found in pathogenic trypanosomatids are discussed, as well as their roles in successful infection. Based on the published genomic and proteomic maps, the panel of trypanosomatid cell death molecules was constructed under different experimental conditions. The lack of PCD molecular regulators and executioners in these parasites up to now has led to cell death being classified as an unregulated process or incidental necrosis, despite all morphological evidence published. In this context, the participation of metacaspases in PCD was also not described, and these proteases play a crucial role in proliferation and differentiation processes. On the other hand, autophagic phenotype has been described in trypanosomatids under a great variety of stress conditions (drugs, starvation, among others) suggesting that this process is involved in the turnover of damaged structures in the protozoa and is not a cell death pathway. Death mechanisms of pathogenic trypanosomatids may be involved in pathogenesis, and the identification of parasite-specific regulators could represent a rational and attractive alternative target for drug development for these neglected diseases.

The double-edged sword in pathogenic trypanosomatids: the pivotal role of mitochondria in oxidative stress and bioenergetics.

The pathogenic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. are the causative agents of African trypanosomiasis, Chagas disease, and leishmaniasis, respectively. These diseases are considered to be neglected tropical illnesses that persist under conditions of poverty and are concentrated in impoverished populations in the developing world. Novel efficient and nontoxic drugs are urgently needed as substitutes for the currently limited chemotherapy. Trypanosomatids display a single mitochondrion with several peculiar features, such as the presence of different energetic and antioxidant enzymes and a specific arrangement of mitochondrial DNA (kinetoplast DNA). Due to mitochondrial differences between mammals and trypanosomatids, this organelle is an excellent candidate for drug intervention. Additionally, during trypanosomatids' life cycle, the shape and functional plasticity of their single mitochondrion undergo profound alterations, reflecting adaptation to different environments. In an uncoupling situation, the organelle produces high amounts of reactive oxygen species. However, these species role in parasite biology is still controversial, involving parasite death, cell signalling, or even proliferation. Novel perspectives on trypanosomatid-targeting chemotherapy could be developed based on better comprehension of mitochondrial oxidative regulation processes.

Vesicles as carriers of virulence factors in parasitic protozoan diseases.

Different types of shed vesicles as, for example, exosomes, plasma-membrane-derived vesicles or microparticles, are the focus of intense research in view of their potential role in cell-cell communication and under the perspective that they might be good tools for immunotherapy, vaccination or diagnostic purposes. This review discusses ways employed by pathogenic trypanosomatids to interact with the host by shedding vesicles that contain molecules important for the establishment of infection, as opposed to previous beliefs considering them as a waste of cellular metabolism. Trypanosomatids are compared with Apicomplexa, which circulate parasite antigens bound to vesicles shed by host cells. The knowledge of the origin and chemical composition of these different vesicles might lead to the understanding of the mechanisms that determine their biological function.

Arginine kinase in Phytomonas, a trypanosomatid parasite of plants.

Phytomonas are trypanosomatid plant parasites closely related to parasites that cause several human diseases. Little is known about the biology of these organisms including aspects of their metabolism. Arginine kinase (E.C. 2.7.3.3) is a phosphotransferase which catalyzes the interconversion between the phosphagen phosphoarginine and ATP. This enzyme is present in some invertebrates and is a homolog of another widely distributed phosphosphagen kinase, creatine kinase. In this work, a single canonical arginine kinase isoform was detected in Phytomonas Jma by enzymatic activity assays, PCR, and Western Blot. This arginine kinase is very similar to the canonical isoforms found in T. cruzi and T. brucei, presenting about 70% of amino acid sequence identity and a very similar molecular weight (40kDa). The Phytomonas phosphagen system seems to be very similar to T. cruzi, which has only one isoform, or T. brucei (three isoforms); establishing a difference with other trypanosomatids, such as Leishmania, which completely lacks phosphagen kinases, probably by the presence of the arginine-consuming enzyme, arginase. Finally, phylogenetic analysis suggests that Kinetoplastids' arginine kinase was acquired, during evolution, from the arthropod vectors by horizontal gene transfer.

Trypanosomatids in phyllostomids (chiroptera, phyllostomidae) from Perobas Biological Reserve, Paraná, Brazil / Tripanossomatídeos em filostomídeos (chiroptera, phyllostomidae) da Reserva Biológica das Perobas, Paraná, Brasil / Tripanossomatídeos en filostomídeos (chiroptera, phyllostomidae) de la Reserva Biológica de las Perobas, Paraná, Brasil

The family Trypanosomatidae reunites a great number of species which parasite several organisms, among them, chiropterans, which may act as reservoirs. The present study aimed at demonstrating the occurrence of trypanosomatids in the blood of phyllostomids from Perobas Biological Reserve, Paraná, Brazil. The capture of the animals was performed with the aid of mist nets in July and August, 2008. The bats were contained manually and for the parasite analysis, a small drop of blood was collected in order to prepare imprint samples which were fixed in absolute methanol. In the laboratory, the slides were stained by the method of Giemsa, observed and photographed with the aid of a light microscope. Fifteen positive results were found out of 33 animals captured. Five of the species captured, Artibeus lituratus, Artibeus fimbriatus, Artibeus planirostris, Artibeus obscurus and Sturnira lilium presented trypanosomatids. Only two species did not have positive slides, Carollia perspicillata and Pygoderma bilabiatum. The species with positive slides have a diversified diet, including the ingestion of insects, which may facilitate the infection. These results contribute with information about the occurrence of these blood parasites in bats since studies about the subject in Brazil are scarce.(AU)

A família Trypanosomatidae reúne grande número de espécies que parasitam os mais diversos organismos, entre eles, os quirópteros, que podem atuar como reservatórios. O presente estudo teve por objetivo investigar a ocorrência de tripanossomatídeos no sangue de filostomídeos da Reserva das Perobas, Paraná. A captura dos animais ocorreu com o uso de redes de neblina, nos meses de julho e agosto de 2008. Os morcegos foram contidos manualmente, e, para a análise dos parasitas, uma gotícula de sangue foi retirada para a confecção do esfregaço e fixação com metanol absoluto. No laboratório as lâminas foram coradas pelo método Giemsa, observadas e fotografadas ao microscópio. Foram obtidos 15 resultados positivos dos 33 exemplares capturados, sendo que cinco das sete espécies capturadas, Artibeus lituratus, Artibeus fimbriatus, Artibeus planirostris, Artibeus obscurus e Sturnira lilium, apresentaram tripanossomatídeos sanguíneos. Somente em Carollia perspicillata e Pygoderma bilabiatum, não foram constatados esses parasitos. Os animais com resultados positivos têm uma dieta bastante diversificada, incluindo a ingestão de insetos, o que pode acarretar infecções, caso alimentem-se de insetos contaminados. Esses resultados contribuem com as informações acerca da ocorrência de parasitas sanguíneos em morcegos, haja vista a escassez de estudos sobre o assunto no Brasil. (AU)

La familia Trypanosomatidae reúne gran número de especies que parasitan los más diversos organismos, entre ellos, los quirópteros, que pueden actuar como reservatorios. Esta investigación buscó la ocurrencia de tripanosomatídeos en la sangre de filostomídeos de la Reserva de las Perobas, Paraná. La captura de los animales ocurrió con el uso de redes de neblina, en los meses de julio y agosto de 2008. Los murciélagos fueron contenidos manualmente, y para el análisis de los parasitas, una gota de sangre fue retirada para fregado y fijación con metanol absoluto. En el Laboratorio las láminas fueron coloreadas por el método Giemsa, observadas y fotografiadas al microscopio. Se alcanzó 15 resultados positivos dos 33 ejemplares capturados, siendo que cinco de las siete especies capturadas, Artibeus lituratus, Artibeus fimbriatus, Artibeus planirostris, Artibeus obscurus y Sturnira lilium, presentaron tripanosomatídeos sanguíneos. Solamente en Carollia perspicillata y Pygoderma bilabiatum, no se constató esos parasitas. Los animales con resultados positivos tienen una dieta bastante diversificada, incluyendo la ingestión de insectos, lo que puede causar infecciones caso se alimenten de insectos contaminados. Esos resultados contribuyen con las informaciones acerca de la ocurrencia de parasitas sanguíneos en murciélagos, dada la escasez de estudios sobre el asunto en Brasil. (AU)

Influence of leishmanolysin-like molecules of Herpetomonas samuelpessoai on the interaction with macrophages.

Monoxenous trypanosomatids usually have an invertebrate as the only host in their life cycles, however, they have been found repeatedly in plants and/or mammals. To succeed in colonizing a vertebrate host, the parasite must quickly adapt to drastic changes in the environment (e.g. temperature), which reflect the conditions found in the insect and mammalian hosts. Leishmanolysin is a metalloprotease ubiquitously distributed in trypanosomatids, playing a myriad of functions. In Herpetomonas samuelpessoai, an insect trypanosomatid, the leishmanolysin-like molecule was implicated in the nutrition and insect adhesion. Herein, we showed that leishmanolysin expression is equally expressed in H. samuelpessoai parasites submitted to insect (26 °C) and mammalian (37 °C) temperatures. Also, the parasites grown in both temperatures interacted at similar rates with macrophages. Finally, we showed that leishmanolysin is involved in crucial steps in the interaction of H. samuelpessoai cells with macrophages, since the treatment with either anti-leishmanolysin antibodies or metalloprotease inhibitor 1,10-phenanthroline significantly reduced the association index. Similarly, the treatment of the macrophages with purified leishmanolysin promoted a powerful reduction in the association index, suggesting the direct involvement of macrophage receptors. These results suggest that H. samuelpessoai leishmanolysin molecules are not modulated by temperature and are involved in the interaction with mammalian cells.

Leishmanolysin-like molecules in Herpetomonas samuelpessoai mediate hydrolysis of protein substrates and interaction with insect.

Herpetomonas samuelpessoai, an insect trypanosomatid, produces a 63-kDa metallopeptidase that has similar biochemical/immunological properties to Leishmania leishmanolysin, a virulence factor that participates in different stages of the parasite life cycle. Herein, we described some biochemical characteristics of the major surface metallopeptidase of H. samuelpessoai that led us to infer some probable functions for this peptidase during the parasite-invertebrate interaction. Gelatin-SDS-PAGE, flow cytometry and confocal fluorescence microscopy provided measurements for the relative levels of surface leishmanolysin-like molecules in H. samuelpessoai. Immunocytochemical analysis demonstrated the presence of leishmanolysin-like molecules on the surface and cytoplasm of the parasite. The surface metallopeptidase was active at a broad spectrum of pH and temperature, showing maximum activity at pH 6.0 at 37 degrees C, and an ability to degrade albumin, hemoglobin, IgG, mucin, casein and gut proteins obtained from Aedes aegypti. This wide substrate utilization might support parasite growth and development. Curiously, H. samuelpessoai cells were able to colonize A. aegypti guts. In an effort to implicate a possible role for the metallopeptidase from H. samuelpessoai, living parasites were treated with different compounds before the interaction with gut cells. The pre-incubation with metallopeptidase inhibitors, phospholipase C or anti-leishmanolysin antibodies promoted a significant reduction in the interaction with guts. Similarly, the pre-treatment of gut cells with purified leishmanolysin-like protein drastically diminished the adhesion process. Furthermore, the expression of surface leishmanolysin in H. samuelpessoai cells was drastically enhanced after passage in A. aegypti. These results suggest the participation of homologues of leishmanolysin in the interaction of H. samuelpessoai with the invertebrate vector.

Interaction of the monoxenic trypanosomatid Blastocrithidia culicis with the Aedes aegypti salivary gland.

Blastocrithidia culicis is a monoxenous trypanosomatid that inhabits mosquitoes. Although its life cycle in these insects has not been described, we recently demonstrated that B. culicis colonizes the Aedes aegypti digestive tract and is also able to reach the mosquito's haemocoel. In this work, we describe the interaction of B. culicis with the A. aegypti salivary gland in vitro and in vivo. Ultrastructural analysis reveals different steps of the invasion process, beginning with the insertion of the B. culicis anterior flagellum into the basal lamina of the gland and ending with the protozoan inside the salivary gland acini compartment. Carbohydrates are involved in the initial adhesion of B. culicis cells to the salivary glands, as demonstrated by protozoan binding inhibition assays and fluorescent lectin labeling of the trypanosomatid-salivary gland interaction. B. culicis is able to survive after incubation in vitro in the mosquito haemolymph, and trypanosomatid binding to salivary glands was confirmed by the injection of radioactively labeled protozoa into the mosquito haemocoel. These results suggest that salivary gland invasion could be part of the B. culicis life cycle in A. aegypti, raising the possibility that B. culicis can be transmitted by these mosquitoes.

Single and concomitant experimental infectionsby Endotrypanum spp. and Leishmania (Viannia) guyanensis (Kinetoplastida: Trypanosomatidae) in the Neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae)

Lutzomyia longipalpis females received single and mixed infections with Endotrypanum and Leishmania. Two biological parameters were analyzed: the percentage of infected females and the distribution of flagellates in the gut of the females. The principal comparisons were performed between (1) two strains of Endotrypanum, (2) cloned versus primary sample of one strain of Endotrypanum, (3) Endotrypanum versus Leishmania guyanensis, and (4) the pattern of flagellates behaviour by optical microscopy in females with single or mixed infection versus the identification of parasites isolated from digestive tracts by isoenzyme electrophoresis. Flagellates of Endotrypanum showed distinct patterns of infection suggesting that there is variation between and within strains. The distribution of Endotrypanum and L. guyanensis differed significantly in relation to the colonization of the stomodeal valve. In co-infection with L. guyanensis, a large number of flagellates were seen to be plentifully infecting the stomodeal valve in significantly more specimens than in females infected by Endotrypanum only. However, the electrophoretic profiles of isoenzymes of parasites recovered from all co-infected specimens corresponded to Endotrypanum. This suggests that the mere correlation sand fly infection-biochemical analysis of isolates may induce parasitological incorrect consideration.

Insights into the role of gp63-like proteins in lower trypanosomatids.

Any actual understanding of trypanosomatids in general requires a comprehensive analysis of the less-specialized species as thorough as our knowledge of the more specialized Leishmania and Trypanosoma. In this context, we have shown by antibody cross-reactivity that purified extracellular metallopeptidases from Phytomonas françai, Crithidia deanei (cured strain) and Crithidia guilhermei share common epitopes with the leishmanial gp63. Flow cytometry and fluorescence microscopy analyses indicated the presence of gp63-like molecules on the cell surface of these lower trypanosomatids. Binding assays with explanted guts of Aedes aegypti incubated with purified gp63 and the pretreatment of trypanosomatids with anti-gp63 antibodies indicated that the gp63-like molecules are involved in the adhesive process of these trypanosomatids to the A. aegypti gut wall. In addition, our results indicate for the first time that the gp63-like molecule binds to a polypeptide of 50 kDa on the A. aegypti gut epithelium extract.

Leishmanolysin (gp63 metallopeptidase)-like activity extracellularly released by Herpetomonas samuelpessoai.

In previous studies, we showed that Herpetomonas samuelpessoai produced a large amount of a surface-located metallopeptidase that presented similar biochemical properties to that of gp63 from Leishmania spp., which is a well-known virulence factor expressed by these digenetic parasites. The present study aims to identify the proteolytic activity released by living H. samuelpessoai cells. In this context, the parasites were incubated in phosphate buffer up to 4 h, and the supernatants were obtained by centrifugation and filtration steps and were then applied on SDS-PAGE to determine the secretory protein profile and on gelatin-SDS-PAGE to identify the proteolytic activity. The results demonstrated that H. samuelpessoai secreted at least 12 polypeptides and an extracellular peptidase of 66 kDa. This enzyme had its activity diminished by 1,10-phenanthroline, EDTA and EGTA. This metallopeptidase was active in a broad spectrum of pH, showing maximum activity at pH 6.0 at 37 degrees C. Casein was also cleaved by this secretory proteolytic enzyme, while bovine serum albumin and haemoglobin were not degraded under these conditions. Fluorescence microscopy and flow cytometry using anti-gp63 antibody against leishmanolysin of L. amazonensis demonstrated the presence of similar molecules on the cell-surface of H. samuelpessoai. Moreover, immunoblot analysis showed the presence of a reactive polypeptide in the cellular extract and in the supernatant fluid of H. samuelpessoai, which suggests immunological similarities between these two distinct trypanosomatids. The zinc-metallopeptidase inhibitor 1,10-phenanthroline was able to inhibit the secretion of the 66 kDa metallopeptidase in a dose-dependent manner, while the phospholipase C inhibitor (p-CMPS) did not alter the secretion pattern. Additionally, anti-cross-reacting determinant (CRD) antibody failed to recognize any secreted polypeptide from H. samuelpessoai. Collectively, these results suggest that the gp63-like molecule was released from the H. samuelpessoai surface by proteolysis instead of phospholipolysis, in a similar mechanism to that observed in Leishmania.

Single and concomitant experimental infections by Endotrypanum spp. and Leishmania (Viannia) guyanensis (Kinetoplastida: Trypanosomatidae) in the neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae).

Lutzomyia longipalpis females received single and mixed infections with Endotrypanum and Leishmania. Two biological parameters were analyzed: the percentage of infected females and the distribution of flagellates in the gut of the females. The principal comparisons were performed between (1) two strains of Endotrypanum, (2) cloned versus primary sample of one strain of Endotrypanum, (3) Endotrypanum versus Leishmania guyanensis, and (4) the pattern of flagellates behaviour by optical microscopy in females with single or mixed infection versus the identification of parasites isolated from digestive tracts by isoenzyme electrophoresis. Flagellates of Endotrypanum showed distinct patterns of infection suggesting that there is variation between and within strains. The distribution of Endotrypanum and L. guyanensis differed significantly in relation to the colonization of the stomodeal valve. In co-infection with L. guyanensis, a large number of flagellates were seen to be plentifully infecting the stomodeal valve in significantly more specimens than in females infected by Endotrypanum only. However, the electrophoretic profiles of isoenzymes of parasites recovered from all co-infected specimens corresponded to Endotrypanum. This suggests that the mere correlation sand fly infection-biochemical analysis of isolates may induce parasitological incorrect consideration.

Protease and phospholipase inhibition protect Veneza zonata (Hemiptera Coreidae) against septicemia caused by parasite trypanosomatid 563DT.

Veneza zonata (Hemiptera Coreidae) is an insect which causes losses in several crops, and it is also an important vector of lower trypanosomatids. V. zonata specimens were collected on rural properties in Londrina, state of Paraná, Southern Brazil. Inoculation of Leptomonas 563DT into V. zonata hemocoel caused insect death within approximately 24 h, with large bacterial proliferation into their hemocoels. Some bacteria which were found in the digestive tract of those insects, such as Escherichia coli, Providencia rettgeri, and Kluyveria ascorbata, were also found in their hemolymph, which suggests that trypanosomatid crossing into hemocoel caused mechanical lesions in the digestive tract that allowed intestinal bacteria to infect the hemolymph, thereby leading to lethal septicemia. In this study we analysed proteolytic activities from the 563DT Leptomonas strain, which is pathogenic for V. zonata, aiming at evaluating the potential use of this Leptomonas strain for the biocontrol of the insect. The proteolytic action was evaluated on cells and on culture supernatants of trypanosomatids. We also evaluated the gelatinolytic activities, the action over natural and synthetic substrates for aminopeptidases, and the action of protease inhibitors during all trypanosomatid growth stages. A significant reduction in the number of insect deaths was observed when Leptomonas 563DT were incubated with inhibitors of proteases and phospholipases before being inoculated into the insects, which suggests that those enzymes are involved in the pathogenic mechanism.

The second messenger cyclic 3',5'-adenosine monophosphate in pathogenic microorganisms with special reference to protozoa.

There is a growing body of information on signal transduction components in microorganisms. Elements of the cyclic 3',5'-adenosine monophosphate signaling system and molecules similar to hormones and receptors have been identified in the majority of prokaryotes and unicellular eukaryotes that have been studied. The presence of ligand- and receptor-like molecules in parasitic microorganisms raises the possibility that these molecules may interact with host communication systems. Adrenergic control of proliferation, differentiation, and infectivity has been described in the flagellate protozoan Trypanosoma cruzi, the etiological agent of Chagas' disease. Interactions between host and parasitic systems could also be antibody mediated, with antigenic cross-reactivity between components of their cAMP-dependent systems. In this review, we discuss these possibilities and summarize the existing data in this area.

Flagellate infections of Brazilian sand flies (Diptera: Psychodidae): isolation in vitro and biochemical identification of Endotrypanum and Leishmania.

Flagellate infections were found in 1,063 of 18,895 sand flies collected in the states of Amazonas, Pará, Rondonia and Acre, Brazil. Infection rates were 13.4% (species group Shannoni); 7.5% (subgenus Nyssomyia); 6.7% (subgenus Lutzomyia series Cruciata); 0.5% (genus Psychodopygus) and 3.1% for other sand flies (various subgenera). Leishmania braziliensis guyanensis and L. mexicana amazonensis were isolated, respectively, from the known vectors, Lutzomyia umbratilis and L. flaviscutellata. Single stocks of L. braziliensis-like and L. mexicana-like organisms were isolated, respectively, from L. whitmani and L. yuilli. Thirty-eight flagellate stocks, isolated by direct culture from sand flies were characterized in detail by morphology in culture, behavior in hamsters and mice and by enzyme profiles. Sixteen stocks from Lutzomyia sp. (Shannoni group) were identified as Endotrypanum schaudinni; 8 stocks from Lutzomyia sp. (Shannoni group) were identified as Endotrypanum sp.; 7 stocks from Psychodopygus ayrozai and P. paraensis were identified as Leishmania sp. previously isolated from the armadillo, Dasypus novemcinctus; 2 stocks of Trypanosoma rangeli were isolated from recently fed Lutzomyia sp. (Shannoni group) sand flies; the remaining 5 stocks from L. umbratilis and L. yuilli could not be identified. Observations suggested that Shannoni group sand flies were the natural vectors of Endotrypanum. Leishmania sp. infections in the man-biting flies P. ayrozai and P. paraensis were restricted to the midgut and associated with recent bloodmeals. Unidentified flagellates in L. umbratilis and L. yuilli were distributed throughout the digestive tract with no trace of bloodmeals.