Isolation and in vitro culture of trypanosomes from Leptodactylus ocellatus from the Atlantic Forest in a new experimental culture medium.

The purpose of this study was to verify the in vitro development of Trypanosoma sp. isolated from Leptodactylus ocellatus frogs under a new protocol using a biphasic medium composed of Novy, McNeal, and Nicolle (NNN) blood agar medium as a solid phase and liver infusion, brain heart infusion, and tryptose (LIBHIT) medium as a liquid phase. Blood forms, collected by cardiac puncture or after the maceration of different organs, were inoculated in culture tubes containing the biphasic medium composed by NNN and LIBHIT. Trypanosomes were observed 4 days postinoculation; most bloodstream trypomastigotes had differentiated into epimastigotes and amastigotes by this time. Trypomastigotes were again observed in older cultures (7 days). Parasites were successfully subcultured for 8 mo in this medium and successfully cryopreserved. The present study provides a new protocol medium for the isolation and culture of anuran trypanosomes.

Antimicrobial activity of Paenibacillus polymyxa SCE2 against some mycotoxin-producing fungi.

AIMS: The purpose of this study was to investigate the antagonistic activity of Paenibacillus polymyxa strain SCE2 against mycotoxigenic fungi and to characterize the antimicrobial compound. METHODS AND RESULTS: Strain SCE2 showed a broad inhibition spectrum against different mycotoxigenic fungi. The crude supernatant obtained from strain SCE2 was filtered with Amicon Diaflo membranes, and the antimicrobial activity was detected in the fraction ranging from 0.5 to 1 kDa. The bioautography of this fraction presented two inhibition zones with both indicator strains (Micrococcus sp. and Aspergillus versicolor), suggesting that more than one substance is produced by SCE2. Based on UV-visible spectral and liquid chromatography/mass spectrometry data, phenazine-1-carboxylic acid (PCA) was characterized as the major compound present in the highest purity active fraction. Drastic alterations in the cytoplasm of A. versicolor were observed by electron microscopy. CONCLUSIONS: One of the antimicrobial substances produced by P. polymyxa SCE2 is PCA. SIGNIFICANCE AND IMPACT OF THE STUDY: The broad antifungal spectrum observed by the compound produced by SCE2 suggests that it has the potential to be used as an alternative or supplementary method to chemical pesticides against mycotoxigenic fungi. This is the first description of a phenazine produced by a member of the genus Paenibacillus.

Antimicrobial activity of Croton cajucara Benth linalool-rich essential oil on artificial biofilms and planktonic microorganisms.

We have previously demonstrated that a linalool-rich essential oil from Croton cajucara Benth presents leishmanicidal activity. In the present study, we demonstrate that this essential oil inhibits the growth of reference samples of Candida albicans, Lactobacillus casei, Staphylococcus aureus, Streptococcus sobrinus, Porphyromonas gingivalis and Streptococcus mutans cell suspensions, all of them associated with oral cavity disease. The purified linalool fraction was only inhibitory for C. albicans. Microbes of saliva specimens from human individuals with fixed orthodontic appliances, as well as the reference strains, were used to construct an artificial biofilm which was exposed to linalool or to the essential oil. As in microbial suspensions, the essential oil was toxic for all the microorganisms, while the purified linalool fraction mainly inhibited the growth of C. albicans. The compounds of the essential oil were separated by thin layer chromatography and exposed to the above-cited microorganisms. In this analysis, the proliferation of the bacterial cells was inhibited by still uncharacterized molecules, and linalool was confirmed as the antifungal component of the essential oil. The effects of linalool on the cell biology of C. albicans were evaluated by electron microscopy, which showed that linalool induced a reduction in cell size and abnormal germination. Neither the crude essential oil nor the purified linalool fraction is toxic to mammalian cells, which suggests that the essential oil or its purified components may be useful to control the microbial population in patients with fixed orthodontic appliances.

Occurrence of Leishmania donovani parasitemia in plasma of infected hamsters.

Intracardiac transfusion of plasma, mononuclear cell fraction and blood of infected hamster donors induced visceral leishmaniasis in normal hamster receptors. At the moment of transfusion, the donors already showed all the typical signs of the disease: ascites, cachexia, as well as splenomegaly and a high parasite load in the spleen and liver. All transfused hamsters developed typical visceral leishmaniasis between 90 and 120 days, indicating that all blood products were infectious. Transfusion of the mononuclear cell fraction induced the highest values of parasitic load (spleen, 766 Leishman Donovan Units (LDU); liver, 2650 LDU), splenomegaly and hepatomegaly (spleen-liver/body relative weight: 1.130 and 6.870, respectively). Animals that received the plasma fraction also developed visceral leishmaniasis, showing similar parasitic load (spleen, 107 LDU; liver, 220 LDU) and spleen-liver/body relative weight (1.005 and 6.35, respectively) than those transfused with whole blood. The finding of typical Leishmania donovani infection in animals transfused with plasma demonstrates the possibility of the extracellular location of parasites, free in this blood fraction deprived of red and white blood cells. Fluorescence-assisted cell sorter analysis (FACS) of plasma showed the presence of particles corresponding in size to amastigotes, which fluoresced strongly with the serum of a patient with Kala-azar (73%), but not with normal serum.

Mucin-like molecules form a negatively charged coat that protects Trypanosoma cruzi trypomastigotes from killing by human anti-alpha-galactosyl antibodies.

In the presence of sialic acid donors Trypanosoma cruzi acquires up to 10(7) sialic acid residues on its surface, in a reaction catalyzed by its unique trans-sialidase. Most of these sialic acid residues are incorporated into mucin-like glycoproteins. To further understand the biological role of parasite sialylation, we have measured the amount of mucin in this parasite. We found that both epimastigote and trypomastigote forms have the same number of mucin molecules per surface area, although trypomastigotes have less than 10% of the amount of glycoinositol phospholipids, the other major surface glycoconjugate of T. cruzi. Based on the estimated surface area of each mucin, we calculated that these molecules form a coat covering the entire trypomastigote cell. The presence of the surface coat is shown by transmission electron microscopy of Ruthenium Red-stained parasites. The coat was revealed by binding of antibodies isolated from Chagasic patients that react with high affinity to alpha-galactosyl epitopes present in the mucin molecule. When added to the trypomastigote, these antibodies cause an extensive structural perturbation of the parasite coat with formation of large blebs, ultimately leading to parasite lysis. Interestingly, lysis is decreased if the mucin coat is heavily sialylated. Furthermore, addition of MgCl2 reverses the protective effect of sialylation, suggesting that the sialic acid negative charges stabilize the surface coat. Inhibition of sialylation by anti-trans-sialidase antibodies, found in immunized animals, or human Chagasic sera, also increase killing by anti-alpha-galactosyl antibodies. Therefore, the large amounts of sialylated mucins, forming a surface coat on infective trypomastigote forms, have an important structural and protective role.

Platelet-activating factor induction of secreted phosphatase activity in Trypanosoma cruzi.

The effects of platelet-activating factor (PAF) on the ecto-phosphatase activity of Trypanosoma cruzi were investigated. Living parasites hydrolyzed p-nitrophenyl phosphate (p-NPP) at a rate of 5.71 +/- 0.37 nmol P(i) mg(-1) min(-1). This ecto-phosphatase activity increased to 8.70 +/- 1.12 nmol P(i) mg(-1) min(-1) when the cells were grown in the presence of 10(-9) M PAF. This effect was probably due to stimulation of the release of the ecto-phosphatase and/or the secretion of an intracellular phosphatase to the extracellular medium, as suggested by cytochemical analysis. Modulation of the ecto-phosphatase activity was also observed when PAF was added during the time course of the reaction. WEB 2086, a competitive PAF antagonist, was able to revert PAF effects when both were used at the same concentration. When PAF was added to a membrane enriched fraction preparation of T. cruzi, no alteration on the phosphatase activity was observed. This result suggests an involvement of intracellular signaling, as PAF was only effective on intact cells. Sphingosine and phorbol-12-myristate-13-acetate (PMA) were then used to investigate a possible involvement of protein kinase C (PKC) with PAF-induced phosphatase secretion. Sphingosine by itself stimulated the secretion of a phosphatase but did not significantly interfere with PAF effects on this enzyme. On the other hand, PMA was able to abrogate PAF-induced release of this phosphatase. These data are highly suggestive of a putative involvement of signal transduction mediated by a ligand of mammalian origin (PAF), through PKC and a specific receptor located on the cell surface of the human parasite Trypanosoma cruzi.

High expression of a functional cruzipain by a non-infective and non-pathogenic Trypanosoma cruzi clone.

We compared a Trypanosoma cruzi clone unable to infect or induce pathology in mice (CL-14), with virulent T. cruzi (Y and CL strains) in terms of cruzipain expression, subcellular distribution and functional activity. Our results showed that (1) intracellular Y amastigotes expressed R1 (carboxy-terminal) and R2 (catalytic) domains concentrated in cytoplasmic vesicles, while CL-14 presented R1 labelling on membrane clusters and R2 in intracellular compartments, (2) CL-14-trypomastigotes presented R1 and R2 staining preferentially on flagellar and cellular membranes, similar to CL, but different from Y strain intracellular labelling pattern, (3) flow-cytometry revealed higher expression of R1 by CL-14-trypomastigotes than virulent strains, but R2 staining similar to CL-trypomastigotes, (4) CL-14-trypomastigotes presented normal cruzipain activity in gelatin gels, but different banding patterns were found in CL-14 versus CL and Y strains. Our data rule out failure in cruzipain expression, activity or subcellular distribution as an explanation for CL-14 biological behaviour, but suggest the expression of a different isoform. These results also cast doubt on the putative role of cruzipain as a target of immunopathological responses, since high levels of functional cruzipain are expressed by a non-pathogenic T. cruzi.

Localization of lectin-binding sites on the surface of Trypanosoma cruzi grown in chemically defined conditions.

The transformation of Trypanosoma cruzi epimastigotes to mammal-infective metacyclic trypomastigotes (metacyclogenesis) can be performed in vitro under chemically defined conditions (TAU 3AAG medium). During this process, changes in the nature of cell surface sugar composition and sugar distribution was evaluated using FITC and gold-labeled lectins and observed by flow cytometry and transmission electron microscopy. The pattern of labeling with the lectins from Triticum vulgaris (WGA), Arachis hypogaea (PNA), Limax flavus (LFA), Canavalia ensiformis (Con-A), and Ricinus communis (RCA-I) significantly changed during the metacyclogenic process. The results obtained are discussed in relation to the role played by T. cruzi cell surface carbohydrate residues on the process of parasite-host cell interaction.

The ultrastructure of the gastrodermis and the nutrition of the gill parasitic Atriaster heterodus Lebedev and Paruchin, 1969 (Platyhelminthes: Monogenea).

The gastrodermis of Atriaster heterodus Lebedev & Paruchin, 1969 (Polyopisthocotylea), a gill parasite from Diplodus argenteus (Valenciennes, 1930), is composed of "U"-shape hematin cells and a connecting syncytium, both having cytoplasmic lamellae. These cells show outgrowths and bent folds which were sent to enclose lumen material. The trapped material was then subjected to endocytosis. The nature of ingested food material was comparatively analyzed by cytochemical and histochemical test. Blood residue were detected in the gut but tests for mucins were negative. No intact erythrocytes were observed in the gut lumen.

Cell surface characterization of amastigotes of Trypanosoma cruzi obtained from different sources.

The present study analyses the morphology and the exposition of surface carbohydrates and the Ssp4 antigen of amastigote forms of Trypanosoma cruzi (Y strain) obtained from three different sources: (a) intracellular, isolated from infected Vero cells 3 days after infection, (b) extracellular, isolated from the supernatant of Vero cells 15 days after infection, and (c) axenic, obtained by incubation of tissue culture trypomastigotes in LIT medium, at 37 degrees C for 4 days. No morphological differences were observed by light microscopy among these amastigotes. Transmission electron microscopy of thin sections showed a thick cell coat easily observed on the plasma membrane of axenic amastigotes. Carbohydrate-containing sites on the surface of the three different amastigotes were analysed using lectins, agglutination assays and flow cytometry. Mannose and/or glucose residues were found on the surface of all populations, but intracellular amastigotes showed the highest number. A small group of cells from the different populations expressed galactose and N-acetyl-glucosamine residues. The presence and distribution of the Ssp4 antigen in the different amastigote populations were evaluated using FITC and gold-labelled antibodies, and observed with an electronic programmable individual cell sorter and transmission electron microscopy. Ssp4 antigen was present on the membrane lining the flagellar pocket and on the cell surface, as well as inside the cytoplasmic vesicles of the host cell. Flow cytometry analysis of different amastigote populations showed that intracellular amastigotes presented the highest percentage of Ssp4-expressing cells.

A novel ecto-phosphatase activity of Herpetomonas muscarum muscarum inhibited by platelet-activating factor.

In the present work ecto-phosphatase activity in Herpetomonas muscarum muscarum has been characterized using live parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 4.27 nmol Pi/mg of protein.min. A pH curve was generated, in which these intact flagellates showed the highest phosphatase activity at pH 6.5. Classical inhibitors for acid phosphatase, such as sodium orthovanadate, sodium tartrate, and ammonium molybdate, were used in the experiments and showed different patterns of inhibition. Lithium fluoride, aluminum chloride, and fluoroaluminate complexes were also tested. Although lithium fluoride and fluoroaluminate complexes were capable of inhibiting the phosphatase activity, aluminum chloride stimulated this enzyme. Cytochemical analysis showed the localization of this enzyme on the parasite surface. This ecto-phosphatase activity was also significantly diminished when the parasites were treated with 10(-6) M platelet-activating factor (PAF), a potent phospholipid mediator that promoted cellular differentiation in this parasite.

Ultrastructure of spermatogenesis of Atriaster heterodus (Platyhelminthes, Monogenea, Polypisthocotylea).

The development of spermatozoa in the Polyopisthocotylea Atriaster heterodus was studied by transmission electron microscopy. Spermatogonia were undifferentiated, with irregular nuclei and little cytoplasm. Primary spermatocytes had sparse chromatin and typical synaptonemal complexes. The nuclear chromatin of secondary spermatocytes was in patches along the nuclear envelope and throughout the nucleoplasm. The complete process of fusion of the early spermatids to a common cytoplasmic mass forming a rosette was elucidated. Nuclei migrated to the center of the mass and changed from round to lamellar or tubular in shape. At the borders of the common cytoplasmic mass, the irregular zones of differentiation had microtubules, mitochondria, nuclei, centrioles, and intercentriolar bodies that give rise to 2 flagella. The spermatozoa presented a continuous row of cortical microtubules surrounding 2 parallels axonemes of the 9+1 type.

Identification of mayaro virus nucleocapsid protein in nucleus of Aedes albopictus cells.

The modifications in the pattern of nuclear proteins of Aedes albopictus cells in response to Mayaro virus infection were analysed early and late after infection. The viral capsid (C) protein of 34 kDa (p34) could be detected in the nuclear compartment 4 h after infection, soon after its synthesis in the cytoplasm. In addition to p34, a group of high molecular weight proteins was also present in this compartment late after infection. The exposition of infected cells to supra optimal temperature of growth modifies significantly the pattern of nuclear proteins. However, the stress condition does not inhibit the transport of p34 to the nucleus. The transport of proteins into nuclei was also followed under "in vitro' conditions by incubating radiolabeled post-mitochondrial extract of infected cells with unlabeled nuclei. Under these conditions, as observed "in vivo', a specific transport of viral C protein and of a group of proteins of high molecular weight to the nuclei was also detected. These results indicate that Mayaro virus infection modifies the nuclear protein pattern in invertebrate cells.

Characterization and cellular distribution of heat-shock proteins HSP70 and HSP60 in Trypanosoma cruzi.

At least eight protein members of HSP70 and three of the HSP60 families have been identified in Trypanosoma cruzi; of these, five HSP70 isoforms and one HSP60 isoform were respectively induced by a 2-hr heat-shock treatment at 37 degrees C. Immunoelectronmicroscopy of epimastigote, spheromastigote, and metacyclic cells obtained in vitro showed anti-HSP60 reactive proteins in the mitochondria and near the kinetoplast. Anti-HSP70 reactive proteins presented a more complex pattern. They were observed in different cellular compartments, and after heat shock all the three cell forms analyzed presented gold particles associated with the cellular membrane.

Effect of platelet-activating factor on cell differentiation of Trypanosoma cruzi.

The effects of platelet-activating factor (PAF), at (10)-6M and (10)-9M, on cell growth and cell differentiation of Trypanosoma cruzi were investigated. Cell differentiation was evaluated by both light and electron microscopy. At the concentrations used, PAF slightly interfered with the protozoan growth. However, parasites growth in the presence of PAF were significantly more differentiated than those grown in the absence of PAF, beginning on the fourth day of culture. A specific PAF receptor antagonist (WEB 2086) totally abrogated PAF effect on cell differentiation. These findings indicate that PAF triggers the process of cell differentiation in T. cruzi and suggest that these parasites have receptors for PAF.

Cell surface analysis of trypanosomes of the subgenus Schizotrypanum isolated from bats.

Two stocks (M5, M29) of trypanosomes of the subgenus Schizotrypanum were isolated from the bat Phyllostomus hastatus and analyzed for cell electrophoretic mobility (EPM) and lectin binding surface sites. Epimastigotes from the M5 and M29 stocks presented a mean EPM of around -0.57 and -0.56 microns. s-1.V-1.cm respectively. Differences in the agglutination profiles were detected between epimastigotes or trypomastigotes from the two parasite populations using lectins with specificity for D-GlcNAc, D-GalNAc, D-Gal and D-Man as probe. Major variation was observed between epimastigote forms. Additionally, the D-GlcNAc binding lectins WGA and BS II strongly interacted with the trypomastigote from both M5 and M29 stocks; this fact is evidence that these trypanosomes are distinct from Trypanosoma (Schizotrypanum) cruzi.

Scanning electron microscopy of adult Wuchereria bancrofti (Nematoda: Filarioidea).

Despite the great importance of Wuchereria bancrofti in causing human lymphatic filariasis, only conventional morphological studies have been completed with adult forms of this filaria. No ultrastructural studies have been carried out, mainly due to the difficulty in obtaining viable parasites from human tissues and the lack of a suitable experimental model or in vitro cultivation. With the recent success in using ultrasound to localize adult worms in living tissues and their surgical recovery from human lymphatic vessels, we show in the present paper the first ultrastructural observations of this filarial form. The cuticle surface analyzed by scanning electron microscopy showed a transversal striated aspect with periodic annulations and many small protuberances irregularly distributed along the filaria. The adults present a specialized cephalic area, with the oral opening surrounded by a circular mouth, papillae, and amphidial opening. Other structures, for example the anal, vulvar, and cloacal opening and spicules, were also observed and are described herein.

Ultrastructural and cytochemical aspects of the cuticle of adult Wuchereria bancrofti (Nematoda: Filarioidea).

Because of the practical limitations of obtaining viable adult forms of the Wuchereria bancrofti, the major species responsible for human lymphatic filariasis, only few ultrastructural studies were carried out. Adult worms present the cuticle as the interface structure between host and parasite. Cuticle structure and the demonstration of the presence of basic proteins, lipids, small amounts of terminal carbohydrate residues, phospholipids and collagen in the cuticle was undertaken on thin sections of embedded parasites. Using immunocytochemical methods, antigenic epitopes similar to those found in the extra cellular matrix of vertebrates were localized on thin sections of the Lowicryl embedded adult filariae.

Cytochemical analysis of the sheath of microfilariae of Wuchereria bancrofti and Brugia malayi.

Thin sections of Epon and Lowicryl embedded microfilariae of Wuchereria bancrofti and Brugia malayi were analyzed by transmission electron microscopy aiming at topochemical characterization of the sheath. Three layers could be distinguished. Some of the layers were labeled when incubated in the presence of antibodies, lectins and enzymes which recognize extracellular matrix components usually associated with the basal laminae lining epithelial cells.

An ultrastructural, cytochemical and freeze-fracture study of the surface structures of Brugia malayi microfilariae.

Ultrastructural analysis of the cuticle of Brugia malayi microfilariae indicated that it is composed of 2 regions: the inner one 15-20 nm thick with a homogeneous aspect and the outer one, designated as epicuticle, which is 15-20 nm thick. Three laminae separated by electron-lucent regions were seen in the epicuticle. Labeling of the cuticle and epicuticle of B. malayi and Wuchereria bancrofti microfilariae was observed when thin sections of Lowicryl-embedded parasites were incubated in the presence of gold-labeled phospholipase-C. Replicas of freeze-fractured microfilariae showed the presence of 2 fracture planes in the epicuticle and no fracture plane in the inner region of the cuticle. The P face of the epicuticle outer fracture plane presented few particles similar to intramembranous particles (IMPs). The epicuticle inner fracture plane P and E faces presented large numbers of densely-packed small particles and many protuberances. Also, fracture faces of hypodermal and muscle cell plasma membranes were analyzed. Faces P and E of fractured membranes showed the presence of typical IMPs. P faces of both membranes showed larger amounts of particles than E faces. Fracture of muscle plasma membrane revealed a linear array of particles disposed in parallel rows on its P face.