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.

If host is refractory, insistent parasite goes berserk: Trypanosomatid Blastocrithidia raabei in the dock bug Coreus marginatus.

Here we characterized the development of the trypanosomatid Blastocrithidia raabei in the dock bug Coreus marginatus using light and electron microscopy. This parasite has been previously reported to occur in the host hemolymph, which is rather typical for dixenous trypanosomatids transmitted to a plant or vertebrate with insect's saliva. In addition, C. marginatus has an unusual organization of the intestine, which makes it refractory to microbial infections: two impassable segments isolate the anterior midgut portion responsible for digestion and absorption from the posterior one containing symbiotic bacteria. Our results refuted the possibility of hemolymph infection, but revealed that the refractory nature of the host provokes very aggressive behavior of the parasite and makes its life cycle more complex, reminiscent of that in some dixenous trypanosomatids. In the pre-barrier midgut portion, the epimastigotes of B. raabei attach to the epithelium and multiply similarly to regular insect trypanosomatids. However, when facing the impassable constricted region, the parasites rampage and either fiercely break through the isolating segments or attack the intestinal epithelium in front of the barrier. The cells of the latter group pass to the basal lamina and accumulate there, causing degradation of the epitheliocytes and thus helping the epimastigotes of the former group to advance posteriorly. In the symbiont-containing post-barrier midgut segment, the parasites either attach to bacterial cells and produce cyst-like amastigotes (CLAs) or infect enterocytes. In the rectum, all epimastigotes attach either to the cuticular lining or to each other and form CLAs. We argue that in addition to the specialized life cycle B. raabei possesses functional cell enhancements important either for the successful passage through the intestinal barriers (enlarged rostrum and well-developed Golgi complex) or as food reserves (vacuoles in the posterior end).

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.

Intestinal NF-κB and STAT signalling is important for uptake and clearance in a Drosophila-Herpetomonas interaction model.

Dipteran insects transmit serious diseases to humans, often in the form of trypanosomatid parasites. To accelerate research in more difficult contexts of dipteran-parasite relationships, we studied the interaction of the model dipteran Drosophila melanogaster and its natural trypanosomatid Herpetomonas muscarum. Parasite infection reduced fecundity but not lifespan in NF-κB/Relish-deficient flies. Gene expression analysis implicated the two NF-κB pathways Toll and Imd as well as STAT signalling. Tissue specific knock-down of key components of these pathways in enterocytes (ECs) and intestinal stem cells (ISCs) influenced initial numbers, infection dynamics and time of clearance. Herpetomonas triggered STAT activation and proliferation of ISCs. Loss of Relish suppressed ISCs, resulting in increased parasite numbers and delayed clearance. Conversely, overexpression of Relish increased ISCs and reduced uptake. Finally, loss of Toll signalling decreased EC numbers and enabled parasite persistence. This network of signalling may represent a general mechanism with which dipteran respond to trypanosomatids.

cAMP signalling in trypanosomatids: role in pathogenesis and as a drug target.

Despite recent research linking cAMP signalling to virulence in trypanosomatids and detailed studies of trypanosomatid adenylyl cyclases (ACs) and phosphodiesterases (PDEs) since their discoveries 40 years ago, downstream components of the pathway and their biological functions have remained remarkably elusive. However, in recent years, significant discoveries have been made: a role for parasite ACs has been proposed in cytokinesis, evasion of the host immune response, and social motility. cAMP phosphodiesterases PDEB1 and PDEB2 were found to be essential for survival and virulence of Trypanosoma brucei and, in Trypanosoma cruzi, PDEC2 was shown to be required for normal osmoregulation. As we discuss here, these breakthroughs have led to an ongoing surge in the development of PDE inhibitors as lead compounds for trypanocidal drugs.

Role of trypanosomatid's arginase in polyamine biosynthesis and pathogenesis.

L-Arginine is one of the precursor amino acids of polyamine biosynthesis in most living organisms including Leishmania parasites. L-Arginine is enzymatically hydrolyzed by arginase producing L-ornithine and urea. In Leishmania spp. and other trypanosomatids a single gene encoding arginase has been described. The product of this gene is compartmentalized in glycosomes and is the main source of L-ornithine for polyamine synthesis in these parasites. L-Ornithine is substrate of ornithine decarboxylase (ODC) - one of the key enzymes of polyamine biosynthesis and a validated target for therapeutic intervention - producing putrescine, which in turn is converted to spermidine by condensing with an aminopropyl group from decarboxylated S-adenosylmethionine. Unlike trypanosomatids, mammalian hosts have two arginases (arginase I and II), which have close structural and kinetic resemblances, but localize in different subcellular organelles, respond to different stimuli and have different immunological reactivity. Arginase I is a cytosolic enzyme, mostly expressed in the liver as a pivotal component of the urea cycle, providing in addition L-ornithine for polyamine synthesis. In contrast, arginase II localizes inside mitochondria and is metabolically involved in L-proline and L-glutamine biosynthesis. More striking is the role played by L-arginine as substrate for nitric oxide synthase (NOS2) in macrophages, the main route of clearance of many infectious agents including Leishmania and Trypanosoma cruzi. In infected macrophages L-arginine is catalysed by NOS2 or arginase, contributing to host defense or parasite killing, respectively. A balance between NOS2 and arginase activities is a crucial factor in the progression of the Leishmania infection inside macrophages. In response to T-helper type 2 (Th2) cytokines, resident macrophages induce arginase I inhibiting NO production from L-arginine, thereby promoting parasite proliferation. Conversely, the response to T-helper type 1 (Th1) cytokines is linked to NOS2 induction and parasite death. Moreover, induction of any of these enzymes is accompanied by suppression of the other. Specifically, arginase reduces NO synthesis by substrate depletion, and N(ω)-hydroxy-L-arginine, one of the intermediates of NOS2 catalysis, competitively inhibits arginase activity. In spite of abundant data concerning arginases in mammals as well their involvement in parasite killing, there are very few papers regarding the actual role of arginase in the parasite itself. This review is an update on the recent progress in research on leishmanial arginase including the role played by this enzyme in the establishment of infection in macrophages and the immune response of the host. A comparative study of arginases from other kinetoplatids is also discussed.

Conservation of the pro-apoptotic nuclease activity of endonuclease G in unicellular trypanosomatid parasites.

Endonuclease G is a mitochondrial protein implicated in DNA fragmentation during apoptosis in cell types ranging from fungi to mammals. Features of programmed cell death have been reported in a number of single-celled organisms, including the human trypanosomatid parasites Leishmania and Trypanosoma. However, the protozoan cell death pathways and the effector molecules involved in such processes remain to be identified. In this report, we describe the pro-apoptotic function of endonuclease G in trypanosomatid parasites. Similar to metazoans, trypanosome endoG showed intrinsic nuclease activity, is localized in mitochondria and is released from this organelle when cell death is triggered. Overexpression of endoG strongly promoted apoptotic cell death under oxidant or differentiation-related stress in Leishmania and, conversely, loss of endoG expression conferred robust resistance to oxidant-induced cell death in T. brucei. These data demonstrate the conservation of the pro-apoptotic endonuclease activity of endoG in these evolutionarily ancient eukaryotic organisms. Furthermore, nuclear DNA degradation by endoG upon release from mitochondria might represent a caspase-independent cell death mechanism in trypanosomatid parasites as genes encoding caspase-like proteins have not been identified in their genomes.

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.

Experimental pathogenicity of a presumed monoxenous trypanosomatid isolated from humans in a murine model.

Two strains of a presumed lower trypanosomatid isolated from immunocompetent and HIV-infected humans in French West Indies were investigated in vitro and in vivo in a murine experimental model. The ability of parasites to grow in vitro in bone marrow-derived macrophages and their virulence in vivo were assessed. For in vivo infection, two groups of BALB/c mice were inoculated either by the subcutaneous or intravenous route with 10(7) promastigotes at day 0. Infection was monitored by measuring parasite load in liver, spleen, foot pad, popliteal, and mesenteric lymph nodes and brain from day 7 to day 150 post-infection using a microtitration technique. Parasites multiplied in mouse macrophages in vitro. In vivo, both strains proved infective to mice and capable of visceralization and dissemination in the popliteal and mesenteric lymph nodes, liver, spleen, and even brain. Both strains elicited a strong humoral response against trypanosomatid antigen in mice, which cross-reacted with Leishmania antigen. Contrasting with the straightforward dissemination of parasites, the infection was strikingly well tolerated by the murine host with no clinical signs and minimal tissue changes around parasitized macrophage infiltrates.

Characterization of a 200 kDa glycoprotein (Cs-gp200) on the pathogenic piscine haemoflagellate Cryptobia salmositica.

The 200 kDa antigenic doublet of the pathogenic haemoflagellate Cryptobia salmositica Katz, 1951, was detected using a monoclonal antibody (MAb-001) in 1-dimensional SDS-PAGE. This antigenic doublet is a glycoprotein since it was susceptible to both protease K and to sodium m-periodate oxidation and is designated Cs-gp200. It has a PI (isoelectric point) value of about 5.5 (using 2-dimensional gel electrophoresis). It migrated faster under reducing conditions than under nonreducing conditions and was partitioned into the aqueous phase in Triton X-114 phase separation. It is a secretory-excretory product since it was detected in a non-protein culture medium with C. salmositica. These results suggest that the Cs-gp200 is a glycoprotein consisting of carbohydrate determinants and conformational polypeptide with internal disulphide bonds. It is a hydrophilic antigen, is a secretory product of the parasite, and plays a important role in antibody elicitation in immunized fish.

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.

The effect of Blastocrithidia triatomae (Trypanosomatidae) on the reduviid bug Triatoma infestans: influence of group size.

Developmental time and mortality in uninfected larvae of the reduviid bug Triatoma infestans and in those infected by feeding a mixture of blood and cysts of the homoxenous trypanosomatid Blastocrithidia triatomae were compared. Larvae were maintained isolated in 77-cm3 (area 9.6 cm2) beakers or in groups of 20, 30, 40, and 50 bugs per 1-liter beaker (area 722 cm2). In uninfected groups, only a minor proportion of isolated bugs showed delayed development, but in groups infected with B. triatomae, additionally, a retardation in groups of 50 larvae occurred. Infected bugs needed more time to complete development in fourth and fifth instar than did uninfected bugs. Mean mortality rates of about 10% in uninfected groups were unaffected by group size. Mortality rates in most groups of infected bugs were about 50%, but in groups consisting of 50 larvae a statistically significant higher mortality rate of 75% was observed. This indicates a subpathological overcrowding stress, increased by the synergistic action of the flagellate.

Relación huésped-parásito en T. rangeli / Host-parasite relation in T. rangeli

Trata la relación huésped-parásito en Trypanosoma rangeli: transmisión del mismo; su desarrollo cíclico en el intestino, hemolinfa y glándulas salivales de algunas especies de triatominos; características de la infección en vertebrados; patogenicidad del parásito en invertebrados. Se observan diferentes grados de patogenicidad en insectos y variación en la conducta de las distintas cepas de Trypanosoma rangeli en triatominos diferentes, que reflejan la interacción de distintos grados de susceptibilidad o de resistencia de los insectos frente a diferentes grados de infección o virulencia del trypanosoma. Se describen los efectos patológicos del mesmo en insectos -los que pueden ser simulados por otros medios-, y su probable relación con el volumen de la hemolinfa: lesiones en todos los insectos con algún grado de infección; alteración del metabolismo y de la muda; y efectos mortales