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MANNOSE-FUCOSE RECEPTOR EXPRESSION ON SKIN FIBROBLASTS PRIMARY CULTURE INFECTED BY LEISHMANIA AMAZONENSIS PROMASTIGOTE FORMS.

Côrte-Real, S., Soeiro, M.N.C., Moreira, F.A., Oliveira, G. M. & Meirelles, M.N.L.

Departamento de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz - FIOCRUZ, RJ.

In man, trypanosomatids of the genus Leishmania reside and multiply in its amastigote forms within mononuclear phagocytes. Parasites responsible for the different types of leishmaniasis evolved the strategy of evading the host immune system by invading monunuclear phagocyte system by receptor-mediated endocytosis. Previous studies have shown the participation of fibroblasts in the initial steps of the cutaneous leishmaniasis both in vivo and in vitro. As the Leishmania surface is particularly rich in carbohydrates, mainly mannose residues, the question arises whether the organism is recognized by a specific receptor on the fibroblasts surface, which could mediate parasite uptake. The aim of the present study was to characterize the presence and the expression of mannose-fucose receptors (MFR) in primary cultures of mice embryos skin fibroblasts (SF) before and after their infection by L. amazonensis promastigote forms. SF cultures were obtained by skin dissociation using 1 mg/ml Collagenase for 1h/37°C. Promastigotes were used to infect SF for 2, 24 and 48h/32°C in PBS buffer at a parasite:host cell ratio of 10:1. For detection of MFR, a neoglycoprotein (FUC-BSA-FITC) was applied employing two different protocols: 50mg/ml neoglycoprotein was added to the cultures before or after 2% PFA fixation. Peritoneal macrophages were used as controls. Mannosyl residues at the parasite and SF cell surface were detected by 100mg/ml Concanavalin-A (ConA-FITC) for 1h/37°C after 2% PFA fixation. Our results revealed an intense mannosyl residues distribution at the parasite and SF surfaces being the intracellular parasites more strongly labeled than the host cell. No significant difference was noticed between normal and infected SF cultures. SF incubated at 4 °C with Fuc-BSA displayed only a faint labeling. On the other hand, previously fixed SF showed that Fuc-BSA was bound in non-infected SF while after the parasite infection a significant decrease in the labeling was observed. Comparing to the signal displayed at macrophage and SF cell surfaces we found that macrophages expressed less Fuc-BSA binding. The addition of 200mM D-mannose blocked the neoglycoprotein labeling. Literature data related to the modulation of MFR during Mf-Leishmania interaction are controversial. Our results showed a down modulation of the MR during 2, 24 and 48 h interaction of primary fibroblast cultures and L. amazonensis promastigotes.

Supported by CNPq, PAPES/FIOCRUZ.

002

SIALIC ACID EXPRESSION AT SURFACE OF AMASTIGOTE AND TRYPOMASTIGOTE FORMS OF TRYPANOSOMA CRUZI.

Meirelles, M.N.L., Soeiro, M.N.C., Garzoni, L., Soares, R., Calvet, C.M., Barbosa, H.S., and ·Vermelho, A.B.

Lab. de Ultra-estrutura Celular, IOC, FIOCRUZ, RJ.,·Depto de Microbiologia Geral, Inst.de Microbiologia, UFRJ,RJ.

Amastigotes are the replicative forms found in mammalian cells during the intracellular life cycle of the Trypanosoma cruzi. Whitin the host cell'cytoplasm they undergo further differentiation to trypomastigote forms which are non dividing forms. Although the tripomastigotes are the classical known infective forms of the parasite, several studies from the literature have shown that the amastigotes can also invade many cell types, including heart muscle cells in vitro ( Meirelles et al.,Mem. Inst. Oswaldo Cruz,Vol. 91, supp, 1996). Sialic acids are acidic monosaccharides usually found at the outermost ends of the sugar chains of animal glycoconjugates. It is well known that T. cruzi expresses a trans-sialidase activity on its membrane surface which speciifically recognizes a2-3 linked sialic acid.

Here we studied the expression of the sialic acid distribution in the two invasive forms of T. cruzi, Y strain, using a panel of three lectins with sialic acid specificity: Maackia amurensis agglutinin a2-3(MAA), Sambucus nigra agglutinin a2-6 SNA) and Limax Flavus agglutinin all Sia linkage (LFA). Isolated amastigote forms were obtained from monolayers of hepatocyte cultures grown in a defined medium supplement with hormones and trypomastigote forms were obtained from the blood of infected mice. Both forms adhered to polylisine glass coverslips, were fixed for 4 minutes at room temperature in 2% paraformaldehyde, rinsed and then incubated for 1 h at 37°C with FITC fluorescent lectins, in a 1/100 dilution. DAPI was used to stain the nucleus.

Amastigote forms surface displayed a strong fluorescente with MAA and SNA lectins and a moderate expression with LFA. Trypomastigote forms showed a stronger reaction with MAA than with SNA and LFA. With the two first lectins we could observe that kinetoplast and nucleous were absent of reaction. With LFA the parasite displayed a very strong reaction in the flagelum. Data from the literature pointed to the presence of sialic acid at the surface of the trypomastigote forms of the parasite. Our results showed differences between the two forms in their membrane surface profile probably indicating different expression of sialic acid and pointing to different manners of these two forms interact with the host cells.

Supported by CNPq, PAPES/FIOCRUZ, PADCT/CNPq.

003

CHARACTERIZATION OF SIALYL RESIDUES DURING THE INTRACELLULAR CYCLE OF TRYPANOSOMA CRUZI IN HEART MUSCLE CELLS.

Meirelles, M.N.L., Soeiro, M.N.C., Garzoni, L., Silva, D. T., Cruz, W.B., Barbosa, H.S. and ·Vermelho, A.B.

Lab. de Ultraestrutura Celular, Instituto Oswaldo Cruz, FIOCRUZ, RJ, ·Depto de Microbiologia Geral, Inst.de Microbiologia, UFRJ, RJ.

Sialic acids (Sia) are a family of about 40 related sugars derived from neuraminic acid which is the most diversely substituted sugar know in nature. It is well established its role in many biological phenomena, especially in the regulation of molecular and cellular recognition phenomena involving carbohydrate binding proteins. T. cruzi express a trans-sialidase enzyme that catalyzes the transfer of bound sialic acids from host glycoconjugates to parasite surface molecules containing terminal b-galactosyl residues. The contribution of host cell sialyl residues for T. cruzi invasion has been demonstrated in assays with sialic-acid deficient mutants of CHO cells. Heart muscle cells (HMC) are negatively charged as the most studied eukaryotic cells in that sialic acid residues are most responsible for 25% of such surface negativeness ( Soeiro et al. J. Microsc. Cytol.Pathol. 26, 121-130, 1994). The present study was carried out to follow the expression of sialyl residues during T. cruzi- HMC interaction with a panel of three lectins with sialic acid specificity: Maackia amurensis agglutinin a2-3(MAA), Sambucus nigra agglutinin a2-6 (SNA) and Limax Flavus agglutinin all Sia linkage (LFA). Normal and T. cruzi infected HMC (Y strain) grown on coverslips were fixed for 4 minutes at room temperature in 2% paraformaldehyde, rinsed and then incubated for 1 h at 37°C with FITC flluorescent lectins, in a 1/100 dilution. DAPI was used to stain the nucleus.

For MAA lectin, normal cells display a stronger reaction than the infected ones. With 24 and 48 h of infection the intracellular parasites were seen showing stronger fluorescence than the host cells. For SNA lectin there was also a more intense reaction in normal cells than in infected ones but only a faint fluorescence was seen in the intracellular parasites. With LFA lectin normal and infected HMC had a moderate positive fluorescence but the parasites'fluorescence was not observed within cells. The structural diversity of sialic acids can determine or alter the specific recognition of sialylated sugar chains by a variety of lectins. Our results confirm data from literature related with the presence of Sia in the host cells. Although Sia is present on the surface of T. cruzi many studies have demonstrate that the parasite is unable to sinthesize Sia. The balance of sialic acid and parasite sialidase may be used to tune many biological events including escape from immune defenses, resistence to lysis, invasion and evasion events. Supported by CNPq, PAPES/FIOCRUZ, PADCT/CNPq.

004

REGULATION AND LOCALIZATION OF b AND a-CARDIAC ACTIN mRNAs IN TRYPANOSOMA CRUZI INFECTED HEART MUSCLE CELLS IN VITRO

Pereira, M.C.S.*, Singer, R.H.** and Meirelles, M.N.L*.

*Laboratório de Ultra-estrutura e Biologia Celular, IOC - FIOCRUZ, RJ, Brazil; and Department of Anatomy, Albert Einstein College of Medicine, Bronx - NY, USA.

The actin cytoskeleton plays an important role in many cellular processes, whereby at least seven different actins, comprising a multigene family, are expressed in both temporal- and tissue-specific manners. We have previously described alterations in the cytoskeleton organization of heart muscle cells (HMC) infected with Trypanosoma cruzi in vitro (J. Submicrosc. Cytol. and Pathol. 25: 559, 1993). Our goal is to understand the molecular basis for the regulation of cytoskeleton proteins mRNAs during HMC differentiation and also after their infection with Trypanosoma cruzi. In the present study we have analyzed changes in the steady-state of the b and a-cardiac actin mRNAs by RNA blotting and in situ hybridization techniques.

Oligonucleotides probes were made to 3´-untranslated region of both actin mRNAs. Total cellular RNA was isolated from normal and T. cruzi infected HMC using the Tri-reagent (Molecular Research Center, Inc.). Agarose gel electrophoresis and Northern Blotting were carried out using standard procedures. For in situ hybridization cells were fixed in 4% paraformaldehyde in PBS and hybridized for 3 h at 37°C in hybridization buffer containing 20ng of the specific probe.

Northern analysis showed that expression of a-cardiac and b-actin genes differed significantly in primary cultures of HMC during myogenesis. Alpha-cardiac actin mRNA levels increased during cell differentiation, while b-actin mRNA levels declined. Not only the expression but also the localization of the actin isoforms mRNAs are distinct. Nonmuscle cells displayed b-actin mRNA signal at the cell periphery, while a-cardiac actin mRNA had a perinuclear distribution in myocytes. Our results with T. cruzi infected cells revealed a reduction of 50% in the a-cardiac actin mRNA expression after 72 h of infection and remained lower in later time course. In contrast, b-actin mRNA levels increased approximately 47% after 48 h of infection. In addition, the in situ reaction revealed a delocalization of the b-actin mRNA signal from the periphery to the perinuclear region. Whether such changes in mRNA levels are mediated by transcriptional mechanisms or by altered mRNA stability or both has not yet been resolved. Further studies will be carried out to elucidate this question.

Supported by CNPq, PAPES/FIOCRUZ, PADCT/CNPq and FIOCRUZ.

005

RE-DISTRIBUTION OF PLASMA MEMBRANE COMPONENTS OF THE MACROPHAGE DURING FORMATION OF LEISHMANIA- CONTAINING PARASITOPHOROUS VACUOLE

Henriques, C. & De Souza. W.

Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofisica Carlos Chagas Filho- Universidade Federal do Rio de Janeiro

Leishmania is an intracellular protozoan that resides in a membrane-bound compartment, the parasitophorous vacuole (PV), which has the ability to fuse with the endocytic system. In the Leishmania life cycle promastigote forms found in the insect vector infect macrophages of the vertebrate host and transform into amastigote forms within the PV. These forms multiply, are released following host cell rupture and are then able to infect other macrophages. In order to study the macrophage plasma membrane components (protein and lipids) involved in the PV formation during Leishmania invasion, the peritoneal macrophage surface was labeled with fluorescent probes- PKH26, DTAF and fluorescein-5- thiosemicarbazide, for lipids, proteins and sialic acid, respectively. Peritoneal macrophages obtained from 6 to 12 weeks male mice were cultivated in round cover slips for 24 hous in DMEM containing 10% FCS at 37⁰ C in a 5% CO₂ atmosphere and labelled with PKH26 (10mM) in glucose isotonic solution for 30 seconds at 4⁰ C, DTAF {5-(4,6- diclorotriazinyl) aminofluorescein}, (0,5 mg/ml) for 30 minutes at 4⁰ C and 3- fluorescein-5-thiosemicarbazide (0,66 mg/ml) at a concentration of 0,5 mM for 30 minutes at 4⁰ C. Thereafter the peritoneal mecrophages were allowed to interact with Leishmania amazonensis promastigotes or amastigotes using a parasite/cell ratio of 10:1 for 15 min at 4⁰ C, for 5 minutes at room temperature to allow Leishmania-macrophage binding and washed with cold Hank's to remove the parasites in excess, before placing the cells to interact in DMEM with 10 % FCS at 37⁰ C in a 5% CO₂ atmosphere for 0 (as control), 15, 30 and 60 minutes. After interaction the cultures were fixed in 4% paraformaldehyde in 0,1 M phosphate buffer (pH 7.2) and observed by confocal-laser scanning microscopy. After labeling of the macrophage surface with DTAF followed by interaction with promastigote and amastigote forms for 15 min stainning of the macrophage surface, of the PV membrane and intravacuolar parasites was observed. After longer incubation times labeling of the parasites was reduced and some cytoplasmic vesicles were labeled. After labeling with PKH 26 followed by interaction with the parasites for 15 min, the macrophage surface was intensely labeled. After prolonged incubation intense labeling of the perinuclear region was observed while the parasites were lightly labeled. After labeling of the macrophage surface with flourescein-5- thiosemicarbazide followed by interaction with the parasites, intravacuolar parasites, especially amastigote forms, were labeled. Labeling of the PV membrane, especially at the contact zone with promastigote, was evident. These observations show that during the process of ingestion of Leishmania by macrophages, with the formation of the parasitophorous vacuole, lipids and glycoproteins of the macrophage plasma membrane are interiorized and are part of the vacuolar membrane and are later on transferred to the intravacuolar parasites.

Supported by PRONEX, FINEP, FAPERJ, CNPq

006

DISTRIBUITION OF THE LIPOPHOSPHOGLYCAN (LPG) DURING THE FIRST STEPS OF LEISHMANIA MAJOR - MACROPHAGE INTERACTION

Mariana Duque-de-Mello and Paulo F. P. Pimenta ^* * To whom the correspondence should be sent.

Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, R.J., CEP 28015-620,Brazil. and * Centro de Pesquisas René Rachou- CPqRR; Fundação Oswaldo Cruz - FIOCRUZ, P. O. Box 1743. Av. Augusto Lima, 1715, Belo Horizonte- MG, CEP 30000, Brazil. Fax(031) 295-3115 E-mail : pimenta@netra.cpqrr.fiocruz.br

Parasites of genus Leishmania are the causative agent of an endemic disease that is spread all over world, the leishmaniasis. This protozoan has two diferent forms in its life cicle: promastigote (flagelate form) found in the insect gut, and amastigote (intracellular form) that lives and multiplies inside the macrophage. Promastigote forms, when inoculated into vertebrate hosts by the insect, infect macrophages. These cells are invaded by the parasites after a recognition process between the protozoan and host cell surfaces. Questions about which molecules of parasite cell suface are involved in this process still remain without answers. It's already known that all in vitro promastigotes are able of attach and to invade the host cell, however just the metacyclic promastigotes, that passed througth a metacyclogenisis process, are infectives. Studies demonstrated that during the metacyclogenesis, modifications occur in the most abundant molecule of parasite surface, the lipophosphogycan (LPG) . In this study we used the antibody 79.3, a LPG marker, to observe the participation and distribuition of this molecule during the initial phase of adhesion and entrance into the macrophage. In immunofluorescence experiments we could observe labelled parasites bound to macrophage surfaces . We compared procyclic (noninfective promastigote) entrance and destruction with the survival and infection maintenance of metacyclics. Next we concentrated our studies on the metacyclic behavior inside macrophages, until to 48 hr after the infection. We used the transmission electron microscopy conjugated with LPG immunolabelling. We observed the increase of LPG content within the flagellar pocket of attached metacyclics on the macrophage surface. This fact suggests that this molecule is secreted during the parasite cell interaction. The LPG molecules were also labelled uniformly all over parasite surface. An important fact was the presence of LPG on the adhesion area of the parasite surface with the host cell plasma membrane. The labelling was intense during all the interaction process, including after the parasite ingestion, on the endocytic vacuolar membranes. After the entrance, the parasite surface also remains labelled with the antibody against LPG. We observed that LPG molecules originated from the parasites, leave the vacuolar parasitophorous inside small cytoplasmatic vesicles that are spreaded for all macrophage cytoplasm. These results show that LPG molecules are involved and have a great importance in the first moments of interaction of promastigotes with the macrophages. Thus, the LPG behavior inside the infected macrophage strongly suggests that this molecule collaborates with the invasion and survival mechanisms that allow the permanence of the parasite inside the host cell.

Supported by CNPq/ FAPERJ/ FENORTE/ PRONEX

This investigation received financial support from the UNDP/ World Bank/ WHO Special Programe Research and Training in Tropical Diseases.

007

INTERACTION OF CRITHIDIA AND HERPETOMONAS SPECIES WITH ANOPHELES GAMBIAE, AEDES ALBOPICTUS AND LUTZOMYIA LONGIPALPIS CELL LINES

Lima, PFN¹, Motta, MCM² & Saraiva, EMB¹

1 - Laboratório de Imunobiologia das Leishmanioses - Depto. de Imunologia, Instituto de Microbiologia, UFRJ.

2 - Departamento de Ultraestrutura e Biologia Celular - Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, RJ.

Homoxenous trypanosomatids are characterized by their development only in one host during all their life cycle, usually an arthropod. These protozoa colonize the intestinal tract of insects and some species also invade the haemocele, but in most cases there is little evidence of pathogenicity to the host. However, some pathological effects of trypanosomatids on insects have been related, as behavioural alterations, disturbances of organ systems and effects on pre-adult development. Some trypanosomatid species harbour endosymbiotic bacteria in the cytoplasm.

In the present study, we investigated the endosymbiont influence in the adhesion of the host protozoa to insect cell lines. Interaction assays between Crithidia deanei (normal and aposymbiotic strains), C. desouzai, C.oncopelti, C.fasciculata and Herpetomonas roitmani and the cell lines of Anopheles gambiae (MOS-55), Aedes albopictus (ALBO) and Lutzomyia longipalpis (LL-5) were made. The insect cells were cultured on glass coverslips and allowed to interact with the parasites at ratio of 1:10. After 1-2 hours at 28^oC, non-adhered parasites were removed and the coverslips were fixed and stained with Giemsa. The percentage of cells with adhered parasites and the mean number of parasites per cell were counted and the association index (AI) obtained by multiplying these two numbers. The AI of C. deanei with MOS-55 was 19 (16% of cells with parasites adhered [cpa] and 1.2 parasites/cell [p/c]), C. desouzai was 52 (31% cpa and 1.7 p/c), C. oncopelti was 68% (40% cpa and 1.7 p/c) and H. roitmani 31 (23% cpa and 1.3 p/c), all endosymbiont containing species. On the contrary, the AI of the endosymbiont-free strains with the different insect cell lines was very low, varing between 1.5 and 4.Assays with LL-5 and ALBO cell lines were similar to the data obtained with MOS-55, were symbiont bearing strains presented higher AI when compared to the aposymbiotic strains.

Transmission electron microscopy showed that the protozoa/insect cell lines interaction usually do not involved the parasite flagellum, but small areas of the cell body. Ocasionally, protozoa were seen in the cytoplasm of the insect cell lines, inside vacuoles. Previous studies have shown that the symbiotic bacteria promotes morphological and physico-chemical alterations in the host cell.Our results reinforce this idea, as the presence of the endosymbiont seems to have an important role in the adhesion of the host protozoa to all insect cell lines.

Supported by: FUJB-UFRJ, CNPq/FIOCRUZ, FINEP & FAPERJ

008

INVOLVEMENT OF LPPG IN THE TRYPANOSOMA CRUZI- INSECT INTERACTION

Nogueira, N.F.S¹, de Souza, W^1,2., Azambuja, P³., Garcia, E³., Mello, C.B.³, Zingales, B⁴. and Colli, W⁴.

¹Centro de Biociências e Biotecnologia, UENF, ²Instituto de Biofísica Carlos Chagas Filho, UFRJ, ³Instituto Oswaldo Cruz, FIOCRUZ, and ⁴Instituto de Química, USP.

During its life cycle in the invertebrate host, Trypanosoma cruzi multiplies as epimastigote forms in the intestine. It has also been shown that these forms attach to the intestine and the rectum wall and that such attachment seems to be important for further transformation of epimastigotes into trypomastigotes. In the present study we analyzed the process of interaction of epimastigotes of T. cruzi with the intestinal epithelium of Rhodnius prolixus in vitro. The insects were used 10 days after blood feeding, when the epithelial cells are covered with the extracellular (perimicrovillar) membranes. The insects were anesthetized , the intestine was removed and longitudinally sectioned, exposing the epithelium which was washed with PBS and allowed to interact for 5 or 30 minutes in the presence of epimastigotes and trypomastigotes of the Y and Dm28 strains. The interaction was followed by interferencial contrast video microscopy. The observations showed that both T. cruzi developmental stages move towards the epithelial surface. However, while trypomastigotes did not attach to the epithelial surface all epimastigotes strongly attached through the flagellum. Scanning electron microscopy showed that such attachment preferentially occurs at the periphery of the cells. Transmission electron microscopy showed that the flagellum of the parasites attach to the extracellular membranes rather than on the plasma membrane of the epithelial cells. Lipopeptidophosphoglycan (LPPG) is a ceramide-containing glycosylphosphatidylinositol compound (GIPL) abundant on the epimastigote plasma membrane in amounts estimated as 1-1.5x 10⁷ molecules per cell. Addition of 20 mM LPPG to the interaction medium (200,000 parasites per 4.5 mm² epithelial tissue) markedly inhibited the binding of epimastigotes to the epithelial surface. Such inhibition was almost complete after short incubation times. Treatment of the LPPG with trifluoroacetic acid, which hydrolyses galactofuranose residues, significantly reduced its inhibitory effect. Intense labeling of the epithelial surface was observed when the intestine was sequentially incubated in the presence of LPPG, polyclonal antibodies recognizing LPPG and fluorescein-labeled goat anti-rabbit IgG, suggesting the presence of a receptor for LPPG on the intestinal epithelial cells. These observations suggest tha LPPG, is involved in the process of parasite-invertebrate host interaction.

This work has been supported by FINEP, FAPESP, CNPq, PRONEX and PAPES (FIOCRUZ).

009

A PHOSPHATIDYLINOSITOL 3-KINASE-MEDIATED PATHWAY INVOLVED IN LEISHMANIA INFECTION

Melo MB; De Souza W; Vannier-Santos MA.

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro.

Phosphatidylinositol (PI) 3-kinases have emerged as important enzymes, coupling diverse extracellular stimuli to intracellular responses. These enzymes have been implicated in vesicular trafficking, secretion and endocytosis. It was seen that infection of host cells by the bacteria Listeria monocytogenes is dependent on PI 3-kinase activity. Here we investigated whether this kinase also mediate Leishmania amazonensis infection. We used wortmannin, a fungal toxin with potent inhibitory effects on PI 3-kinases, to verify the importance of PI 3-kinase-mediated events during parasite entry. Pre-treatment of either mouse peritoneal macrophages and promastigote forms with wortmannin at a nanomolar range caused significant reduction in the infection rates. Ultrastructural analysis of wortmannin-treated L. amazonensis promastigotes revealed several alterations in diverse cellular structures. Cells presented numerous volutin granules at different stages of formation and amorphous sickle-shaped deposits were observed within membrane cisternae resembling the endoplasmic reticulum. An increase in lipid inclusions was also observed, suggesting a lipid metabolism dysfunction. Remarkable alterations were also seen in the Golgi apparatus. Frequently the trans cisternae presented an unusual curvature, culminating sometimes in the formation of vesicle-like compartments, suggesting the participation of PI 3-kinase in the parasite vesicular trafficking. Taken together these data indicate that PI 3-kinase activity is required not only to regulate the macrophage phagocytic function but also for a mechanism that parasites employ to promote their uptake.

Supported by FAPERJ, FUJB, TWAS, CNPq, CEPG-UFRJ and PRONEX.

010

INDUCTION OF MACROPHAGE TYROSINE DEPHOSPHORYLATION UPON INFECTION BY LEISHMANIA AMASTIGOTES

Martiny A¹, Saad-Nehme J², Meyer-Fernandes JR², De Souza W¹, Vannier-Santos MA¹.

¹ Instituto de Biofísica Carlos Chagas Filho and ² Dep. de Bioquímica Médica, Universidade Federal do Rio de Janeiro.

Activation of protein tyrosine kinases (PTKs) is an early step on the signal transduction of extracellular stimuli such as pathogen adhesion. We have previously reported that macrophage infection by Leishmania promastigotes is highly dependent on tyrosine phosphorylation (Martiny et al., Eur J. Cell Biol., 71: 206-215, 1996). Phosphotyrosine levels are modulated by the coordinate action of PTKs and protein tyrosine phosphatases (PTPases). In this regard, bacteria of the genus Yersina express an extremely potent tyrosine phosphatase (YopH) which is translocated to the host cell cytoplasm and dephosphorylates target proteins blocking phagocytosis. We approached the possibility of a similar mechanisms during Leishmania amazonensis amastigote infection. Stimulated, uninfected macrophages presented three major tyrosine phosphorylated proteins of 200, 120 and 60 kDa. The 120 and 60 kDa proteins and a minor phosphorylated doublet of 44-42 kDa were remarkably dephosphorylated after amastigote infection. Amastigote forms present an ecto-phosphatase activity (La-PTPase) which dephosphorylates substrates at tyrosine residues and is sensitive to sodium orthovanadate. Neither vanadate- nor phosphotyrosine-treated parasites produce the dephosphorylation of phagocyte proteins. In addition, macrophages which were treated with vanadate prior to infection also presented a low phosphorylation level, suggesting that parasite phosphatase may have a pivotal role in dephosphorylation. A Western blotting time-course assay revealed that maximum dephosphorylation was achieved after a 60-min infection and was maintained for at least 7 days. Impairment of phagocytosis by cytochalasin D treatment was not able to abolish dephosphorylation, suggesting that amastigotes exert their effect even outside the host cell (Martiny et al., submitted). Nevertheless one cannot discard the possibility that phagocyte phosphatases also take part on the infection process. We are currently identifying these phosphatases and their substrates.

Supported by FAPERJ, FUJB, TWAS, CNPq, CEPG-UFRJ and PRONEX.

PROTEIN KINASE C AND PI-3-KINASE ARE INVOLVED ON THE CONTROL OF MACROPHAGES INVASION BY TRYPANOSOMA CRUZI

Dutra, J. M. F. ¹, Vieira, M. C. F.¹, Carvalho, T. M.. U. ^1,2 and De Souza, W^1,2.

1 Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro. CCS - Bloco G, Cidade Universitária, Ilha do Fundão, Rio de Janeiro - RJ. CEP: 21949-900. 2 Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense. Av. Alberto Lamego 2000, Campos dos Goytacazes, RJ. CEP: 28015-620.

We showed previously the involvement of serine/threonine phosphatases during the internalization process of trypomastigote forms of T. cruzi by peritoneal macrophages and the association of serine/threonine phosphorylated residues with concentrations of F-actin at the contact region between parasites and macrophages (Vieira et al. 1995. Mem. Inst. Oswaldo Cruz., Rio de Janeiro, 90, Suppl. I: 95). The involvement of isoforms of protein kinase C in macrophage phagocytosis has been largely demonstrated (Blystone et al. 1994. J. Cell Biol. 127: 1129-1137; Allen & Aderem 1995. J. Exp. Med 182:829-840; Karimi & Lennartz 1995. J. Immunol. 155: 5786-5784; Zhu et al. 1995 J. Biol. Chem. 270: 17652-17655; Buchwalow et. al., 1997. Acta Histochem. 99: 63-70). In order to verify if these proteins could be involved in the generation of phosphorylated residues during the internalization of trypomastigotes into peritoneal macrophages we previously treated macrophages with calphostin C, a specific inhibitor of protein kinase C, before the interaction with trypomastigotes. This treatment induced a dose dependent inhibition of parasite internalization concomitant with an elevation in the number of attached parasites. This observation suggests that the internalization of trypomastigotes is inhibited under this condition, but the association with macrophage surface was not affected. We also tested the effect of previous treatment of macrophages with wortmmanin, an inhibitor of PI-3-kinase, which is also involved in macrophage phagocytosis (Nobukazo et al. 1996. J. Cell Biol. 135: 1249-1260). The inhibition of macrophages' PI-3-kinase induced a dose - dependent decrease of both internalization and adhesion of the parasites. This effect suggests that, somehow, when PI-3-kinase is inhibited in macrophages the initial recognition between trypomastigotes and these cells is affected. Taken together these data indicate that protein kinase C and PI-kinase play some role on the control of macrophage cellular events that led to internalization of trypomastigote forms of T. cruzi into peritoneal macrophages

Supported by: CNPq, FINEP and PRONEX.

012

ANTIGENS OF TRYPANOSOMA CRUZI TAKEN UP FROM THE CULTURE MEDIUM BY RESIDENT MACROPHAGES

Siqueira,P.L.A., Ribeiro, D.F. & Souto-Padrón,T.

Laboratório de Protozoologia I , Instituto de Biofísica Carlos Chagas Filho - UFRJ - CCS, Bloco G - Ilha do Fundão, Rio de Janeiro, RJ, 21949-900 - Brasil.

Trypanosoma cruzi is an obligate intracellular parasite presenting in mammalian host two evolutive forms : nonreplicative trypomastigote forms that circulate in the blood and amastigote forms that are capable of replication inside the infected cells.Immunity to T. cruzi has focused on mechanisms capable to recognizing and killing trypomastigotes and the presentation of parasite's proteins on the cell surface of infected cells. Previous studies showed the presence of T. cruzi antigens on the cell surface of infected and uninfected host cells (Abrahamsohn & Kloetzel, Parasitology 80:147,1980; Araujo, J.Immunol.. 135:4149,1985; Dias Vieira & Souto-Padrón, Mem.Inst.Oswaldo Cruz 90 Suppl. pp89,1995). This study investigates the interiorization and delivery of T. cruzi antigens by peritoneal macrophages. Brieflly, resident peritoneal macrophages cultivated for 24 hs in DMEM supplemented with 10 % fetal calf serum were incubated in the presence of gold-labeled albumin (10 nm) during 1 h at 37⁰ C and next in the presence of cell culture supernatant fluid obtained after completion of the first intracellular cycle and rupture of infected cells (conditioned medium) for 30 or 60 minutes. After incubation with albumin and conditioned medium, macrophages were fixed in 0.1% glutaraldehyde,4% formaldehyde, 0.5% picric acid in 0.1 M cacodylate buffer, pH7.2, containing 3.7 % sucrose, during 1 h at room temperature. Samples were then dehydrated in ethanol at 4⁰ C and embedded in Unicryl. Thin sections were incubated in the presence of antibodies against SAPA (shed accute phase antigen), cruzipain and LPPG and then in the presence of a gold-labeled goat-anti rabbit antibody (5 nm). One hour after incubation in the presence of gold-labeled albumin a large number of gold particles was observed inside several cytoplasmic vacuoles resembling late endosomes and lysosomes. Thirty minutes after incubation with the conditioned medium T. cruzi SAPA, cruzipain and LPPG were visualized on the macrophage plasma membrane or inside small vacuoles located at the macrophage periphery. One hour after incubation with the conditioned medium, parasite's antigens were seen in the same places as described before and inside the same vacuoles containing gold labeled albumin. We are currently investigating the kinetcs of interiorization of the different antigens, the nature of the different compartments involved in this and the association with MHC molecules.

Supported by PRONEX, FINEP, CNPq & CEPG-UFRJ

013

TRYPANOSOMA CRUZI INFECTION MODULATES THE SYNTHESIS OF ALPHA-2- MACROGLOBULIN RECEPTOR (LRP)

Soeiro, M.N.C.¹, van Leuven F.², Paiva, M.M.¹, Waghabi, M.¹, Coutinho, C.M.L.M.^1,3, Meirelles, M.N.L.¹ and Araújo-Jorge, T.C.¹

¹Depto. Ultra-estrutura e Biologia Celular, IOC, FIOCRUZ, Riode Janeiro, RJ; ²Department of Human Biology, University of Leuven, Leuven, Belgium; ³Depto. Biologia Celular e Molecular, Universidade Federal Fluminense, Niterói, RJ.

Alpha-2-macroglobulin (A2M) is a large glycoprotein (720Kda), acute phase reactant in rodents, that binds and inhibits endoproteinases and some peptides. After interacting with proteinases or methylamine, A2M molecule (F-A2M) suffers a conformational change that allows the exposure of neoantigen sites including a receptor binding domain, being rapidly removed from the circulation by a A2M receptor (A2MR/LRP).

The aim of our study was to evaluate by Northern blots and by immunocytochemical approaches the synthesis and the distribution of A2MR/LRP in the heart and liver of C57/bl6 mice during the acute phase of Chagas' disease. The organs were removed after 1, 7, 14 and 21 days post infection (7.5x10 3 trypomastigotes of the Y strain). The parasitaemia usually occurred between the 7-9 day post infection. For the Northern analysis, total RNA extraction was performed by the guanidinium isothiocyanate method and 10mg/ml of the purified RNA was separated by denaturing agarose gel electrophoresis. After capillary transfer the nylon membrane was hybridized with LRP cDNA (1.4kb EcoRI fragment corresponding to position 776-2197 - van Leuven et al, 1993, Biochim. Biophys. Acta 1173, 71-74) radiolabeled probe, autoradiographs were scanned densitometrically and the values were normalized to the signal obtained with bactin cDNA probe. To detect "in situ" A2MR/LRP, frozen tissues sections were incubated with 50mg/ml F-A2M-FITC for 1 hour at 37ºC. Negative controls were performed by adding 5mM EDTA in the incubation medium, since the A2M binding to its receptor is dependent of Ca²⁺ presence.

The expression of the 15kb A2MR/LRP mRNA was characteristically detected in non infected C57/bl6 liver and heart samples. However, in both organs, mRNA levels displayed a dramatic decline during mice chagasic acute phase. In the liver extracts, a decrease of about 40% and 70-90% of mRNA was detected 1 and 21 days post infection, respectively. In the heart samples A2MR/LRP mRNA expression displayed a stronger decreasing during T. cruzi infection reaching levels of 90% as early as 1 day post infection which was maintained until 21 days post-infection. The analysis of F-A2M-FITC binding in the heart of non infected mice showed a strong labeling at the endothelium vessels but no significant labeling could be seen at the muscle cells. However, in the infected heart tissue (7 days post-infection) less F-A2M-FITC binding was detected at the endothelium, but the labeling could be found in small areas of the myocardium fibers, possibly associated to the parasite nests.

Supported by PAPES/FIOCRUZ, CNPq, PADCT/CNPq and FIOCRUZ.

014

INTERACTION OF TRYPANOSOMA CRUZI TISSUE CULTURE AND BLOODSTREAM TRYPOMASTIGOTES WITH VERO CELL

Toma, H.K.¹ & Romanha, A.J.²

1- Depto de Análises Clínicas, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21949-900, Rio de Janeiro, RJ; 2- Centro de Pesquisas René Rachou, FIOCRUZ, Av. Augusto de Lima, 1715 - C.P. 1743, 30190-002, Belo Horizonte, MG, Brasil.

In vitro studies of T. cruzi-host cell interaction have shown that differences in cell infection depend of cell line, parasite strain and the trypomastigote origin. Trypomastigotes obtained from distinct origins may have qualitative and quantitative differences in surface sites recognized by host cells. In this work, the interaction of T. cruzi tissue culture (TCT) and bloodstream trypomastigotes (BT) with Vero cell was compared. The T. cruzi strains SC-1 and SC-28 from Santa Catarina State and, the reference Y and CL strains were used. The BT were obtained from experimentally infected albino mice and TCT were obtained from previously maintained infected Vero cells. Cells were infected in a 10:1 parasite/cell ratio for 3 h, washed and incubated with DMEM + 10% FBS for 24 and 72 h, then fixed with methanol, Giemsa stained and examined under microscope. The percentage of infected cells and the average number of parasite per cell was determined by counting 500 cells at random. The percentage of cells infected: a) with TCT varied from 15.2 to 37.3% at 24h and from 2.0 to 40.2% at 72h and b) with BT, varied from 0 to 2.0% at 24h and from 0 to 1.0% at 72h. TCT were more infective than BT suggesting that significant alterations in the parasite membrane may occur. Maybe the presence of host elements in BT membrane, such as immunoglobulins or other molecules, may interfere in the interaction with the host cell. The mean of parasite/cell infected: a) with TCT, varied from 1.8 to 2.5 at 24h and from 17.4 to 23.7 at 72h and b) with BT, varied from 0 to 2.0 at 24h and from 0 to 52.0 at 72h. After 72 h, the mean of parasite/cell was the same in cells infected with TCT and BT for SC-1 and SC-28 strains. However, for Y strain, the parasite mean was higher with BT than with TCT infected cells. Although the BT from Y strain infected initially only 2.0% of cells, they presented the highest intracellular parasitism at 72h (52 parasites/cell). Differently from TCT, the BT from CL strain were not able to infect the cells after 3 h contact. These results reinforce the importance of the T. cruzi strain as well as the trypomastigote origin on the parasite-host cell interaction.

Supported by CNPq and FIOCRUZ

015

EFFECT OF HUMAN TYPE I INTERFERONS ON THE GROWTH OF TRYPANOSOMA CRUZI IN HUMAN FIBROSARCOMA CELLS

Cerávolo¹ , I.P.; Totini¹, D.A.; Carvalho², A.F.; Kroon², E.G.; Ferreira², P.C.P.; Romanha¹, A.& Golgher¹, R.R.

1- Centro de Pesquisas René Rachou, FIOCRUZ, Caixa Postal 1743, 30190/002, Belo Horizonte, MG. 2- Departamento de Microbiologia, ICB, UFMG, Caixa Postal 486, 31270/901 Belo Horizonte, MG.

Interferons (IFNs) are cytokines that can inhibit the intracellular growth of viruses, bacteria, fungi and protozoa. It has been shown that Trypanosoma cruzi intracellular development is not affected by type I (a and b) IFNs. However, T. cruzi associates poorly with murine cells when pretreated with murine type I (a/b) IFN. In vivo, the parasitemia is reduced when mice received this IFN. Type II (g) murine IFN activates macrophages to inhibit the multiplication of T. cruzi. So far, these studies were done with impure IFN preparations. Only after the recombinant murine g IFN became available the role of IFNs in the mouse infection by T. cruzi could be finally established. With regard to human type I IFNs, their effects remain to be evaluated with purified preparations. Therefore, in this work, we have investigated if highly purified or recombinant IFNs would influence the intracellular development of T. cruzi. Cells derived from a human fibrosarcoma(2C4) were grown in chamber slides to semi-confluency, treated for 24 h with IFN concentrations that gave a strong antiviral effect and infected with T. cruzi (Y strain) for 6 h. Non-adherent parasites were removed, fresh medium with IFN was added and the cells reincubated for 48 h, when they were fixed and stained with May Grünwald-Giemsa. The percentage of infected cells and the average of parasites per 30 cells was determined to evaluate the effect of the IFNs. Results showed that recombinant a2 and a mutated b (with a serine replacing cysteine at position 17- bser - P2) did not alter the growth of the parasite. A recombinant b IFN, with the natural sequence but with a residue of six histidines added to the NH2 end, (brecV) and two highly purified IFNs derived from the amniotic membrane- the b-like (B9) and another protein fraction (E4) displayed a small inhibiton. Studies are underway to see if these IFNs will show an increased inhibition in human macrophages.

Work supported by CNPq, FAPEMIG, FIOCRUZ. IPC has a studentship from CAPES. RRG is a visiting scientist of the CNPq-FIOCRUZ Agreement. DAT was supported by the CNPq.

016

CYTOCHALASIN D INHIBITS TRYPANOSOMA CRUZI INFECTION IN DIFFERENT HOST CELLS

¹ Rosestolato, CTF; ¹De Souza,W; & ^1,2Carvalho, TMU^1,2

¹Laboratório de Biologia Celular e Tecidual, CBB,UENF

²Laboratótrio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho,UFRJ

Trypanosoma cruzi is a flagellate protozoan that belongs to the Trypanosomatidae family and is the ethiologic agent of Chagas'disease. It can invade and develop within most mammalian cells. After invasion, the trypomastigote form is localized within an endocytic vacuole in both phagocytic and non-phagocytic cells. The mechanism by which trypomastigote invades host cells is controversial. There are discussions if the parasite enters the host cell through an active process or by phagocytosis. So we decided to address this question using different host cell types treated with cytochalasin D and to analyse, using confocal laser scanning microscopy,the distribution of actin filaments of the host cell at the interacting site. The following cell types were used: Vero, LLCMK2, macrophage, L6 and HFS-F. These cells were cultivated and allowed to interact with trypomastigotes (1 cell:10 parasites), for 1 hour at 37^oC in the presence or absence of cytochalasin D (5ug/ml), to depolymerize actin filaments. We made also a control incubating the cells with DMSO (1%). After 1 hour of interaction the cells were washed three times to remove non adherent parasites and processed as follows: a) fixed with Bouin for 5 minutes, stained with Giemsa for 30 minutes, dehydrated in acetone/xylol and mounted in enthelan, observed and counted using a light microscope: b) fixed with 2.5% Glutaraldehyde in 0.1M phosphate buffer pH 7.2 for 1 hour, dehydrated in acetone, critical point dryed with C0₂ , coated with gold and observed in a Scanning Electron Microscope; c) fixed with 0.1% glutaraldehyde, 4% formaldehyde, 0.1% Triton X-100 in PHEM buffer pH6.9 for 3 minutes, washed in the same buffer and incubated in 1:100 phaloidin-FITC for 1 hr and observed in a Confocal Laser Scanning Microscope. Observations by ligth microscopy showed a reduction of the endocytic index and an increase in adhesion index in all tested cells treated with cytochalasin D. Observations by Scanning Electron Microscopy confirmed the results obtained by ligth microscopy in relation to adhesion of trypomastigotes to the host cell. Observations of phaloidin-FITC labeled cells showed two patterns: a) a concentration of actin filaments around trypomastigote forms in close association with host cells, mainly obseved in macrophages, and b) some partialy internalized trypomastigote forms showed no concentration of actin filaments around them.

This study was supported by CNPq, FENORTE, FAPERJ and PRONEX.

017

MANNOSE RECEPTORS LOCALIZED AT THE SURFACE OF HEART MUSCLE CELLS ARE INVOLVED IN THE BINDING AND UPTAKE OF TRYPANOSOMA CRUZI

Soeiro, M.N.C., Barbosa, H.S., Meirelles, M.N.L., Paiva, M.M., Waghabi, M., Guimarães, E.V. and Araújo-Jorge, T.C.

Depto. Ultra-estrutura e Biologia Celular, IOC, FIOCRUZ, Av. Brasil 4365, RJ, Brazil

Carbohydrate-protein interactions play a important role in a wide variety of biological and pathological events. Mannosyl residues and their counter-receptors play importance during the interaction of Trypanosoma cruzi and host cells. We analysed the mannose receptors (MR) in primary cultures of heart muscle cells (HMC) before and after their interaction with trypomastigote forms of T. cruzi. The role of mannosyl residues and MR during this interaction were tested by carbohydrate addition during the HMC-parasite uptake. Ultrastructural studies were performed using horseradish peroxidase coupled to colloidal gold particles (HRP-Au) as probe for MR detection, incubating the cells with HRP-Au for 30min at 4 ºC or 4 h at 37ºC before or after 24 h of interaction with T. cruzi. Infection rates were analysed by the addition of 10 or 50mM of D-mannose during the infection (5:1 parasite\HMC ratio) in Eagle's medium without serum for 24 h at 37ºC. After the host cell-parasite contact the cultures were fixed in Bouin, stainned with Giemsa and the percentage of infected HMC was calculated. Our ultrastructural results showed the presence of the tracer all over the sarcolemma of non-infected HMC mainly near and inside uncoated invaginations, caveolae and a significant labeling at extracellular matrix sites. After 4 h HRP-Au particles were found inside endosomal compartiments displaying different sizes and densities. Infected cultures could be seen with extracellular parasites attached to HMC through HRP-Au particles. The addition of D-mannose to the interaction medium blocked the parasite uptake by HMC being the concentration of 10mM the most effective, reaching levels of 90% of inhibition. On the other hand, the previous parasite treatment did not produce any significant difference. Our present data suggest that mannosyl residues localized at the parasite surface were recognized by lectin-like molecules on the HMC sarcolemma mediating, at least in part, the recognition process between HMC and T. cruzi. The present data are in agreement with our previous biochemical results showing that HRP binding at HMC surface is impaired after T. cruzi infection and that it could be restored by the treatment of the infected cultures with Nifurtimox.

Supported by CNPq, PAPES/FIOCRUZ, PADCT/CNPq and FIOCRUZ

018

LECTIN BINDING ANALYSIS IN TRYPANOSOMA CRUZI-INFECTED AND NON INFECTED- HEART MUSCLE CELLS

Soeiro, M.N.C., Barbosa, H.S., Meirelles, M.N.L., Oliveira, G.M. and Araújo-Jorge, T.C.

Deptº Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, FIOCRUZ, 21045-900, RJ, Brazil

The reversible carbohydrate-lectin molecular interactions are involved in different biological processes as immunity, organogenesis, fertilization and parasite infections. Regarding to Trypanosoma cruzi-host cell interaction many glycoconjugates are putative candidates for their initial molecular recognition. As the heart is one the most affected organ during Chagas' disease, an "in vitro" lectin screening of heart muscle cells (HMC) before and after their infection with trypomastigote forms of T. cruzi was investigated. We started our studies by analysing through immunofluorescence the binding of Wheat-germ agglutinin (WGA), Peanut agglutinin (PNA) and Ricinus communis (RCA) using two different protocols. In both assays, HMC were infected with trypomastigotes for 24 hours (5:1 parasite/host cell ratio) and afterwards were directly incubated for 1 hour at 4ºC with 100mg/ml lectin and immediately fixed with 2% PFA or incubated for 1 hour at 37ºC with the same lectin concentration after 2% PFA fixation. The carbohydrate specificity was evaluated by the addition of 100mM the corresponding carbohydrate during the lectin incubation.

The lectin binding at 4ºC displayed quite different results from those observed when the lectin was added to the previously fixed HMC. PNA and RCA-120 (lectins that specifically bind to b—D-galactosyl residues) binding at 4ºC in both infected and non-infected HMC showed a similar positive pattern with a patchy distribution being more intense at 4ºC than at 37ºC. Confocal analysis showed that PNA labeling at 37ºC with previously fixed HMC displayed a perinuclear distribution. WGA, a lectin that recognizes sialic acid and N-acetyl glucosamine residues, strongly bound to non-infected HMC in similar pattern in both protocols. This lectin was the one with the higher signal when compared to the other studied lectins. Confocal analysis demonstrated that in both protocols most labeling was localized at the sarcolemma. WGA binding to the surface of T. cruzi-infected HMC displayed a lower labeling while the parasitophorous vacuole was positively labeled. The addition of specific carbohydrate efficiently blocked the lectin binding.

Confocal and ultrastructural studies are being done enmploying lectins with other specificities in order to better understand the dinamic events of the recognition process between HMC and Trypanosoma cruzi.

Supported by PAPES, FIOCRUZ, CNPq and PADCT/CNPq.

019

INFECTIVITY OF TRYPANOSOMA CRUZI STRAINS IS ASSOCIATED WITH DIFFERENTIAL EXPRESSION OF SURFACE MOLECULES DISPLAYING DIFFERENTIAL Ca²⁺ SIGNALING ACTIVITY

Rita C. Ruiz*, Silvio Favoreto Jr.*, Patricio M. Manque* Miriam L. Dorta*, Maria E.M. Oshiro^# , Alice T. Ferreira^#, & Nobuko Yoshida*

* Depto. de Microbiologia, Imunologia e Parasitologia, # Depto. de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, R. Botucatu, 862, São Paulo, S.P., Brasil

Metacyclic trypomastigotes of different T. cruzi strains may vary significantly in their ability to enter mammalian cells. What determines such variability is not known. We invetisgated the possibility that the differential Ca²⁺ signaling activity of parasite surface molecules, differentially expressed in T. cruzi strains, determines infectivity. Mammalian cell invasion assays, using G and CL metacyclic trypomastigotes, showed that CL strain enteres target cells in several fold higher numbers as compared to the G strain. Analysis of expression of surface glycoproteins in metacyclic forms of the two strains by iodination, immunoprecipitation and flow cytometry, revealed that gp90, undetectable in CL strain, is one of the major surface molecules in G strain, expression of gp82 is comparable in both strains and gp35/50 is expressed at lower levels in CL strain. Purified gp90 and gp35/50 bound more efficiently than gp82 to cultured HeLa cells. However, the intensity of Ca²⁺ response triggered in HeLa cells by gp82 was significantly higher than that induced by gp35/50 or gp90. Most of Ca²⁺ signaling activity of metacyclic extract towards HeLa cells was due to gp82 and was inhibitable by gp82-specific MAb 3F6. Ca²⁺ mobilization was also triggered in metacyclic trypomastigotes by host cell components, it was mainly gp82-mediated and more intense in CL than in G strain. We propose that expression of gp90 and gp35/50 at high levels impairs binding of metacyclic forms to host cells through the productive gp82-mediated interaction, which leads to target cell and parasite Ca²⁺ mobilization required for invasion. Analysis of metacyclic forms of 8 additional T. cruzi strains corroborated the inverse correlation between infectivity and expression of gp90 and gp35/50.

Supported by FAPESP, CNPq/PADCT.

020

STABILITY OF ADHESION OF TRYPANOSOMA CRUZI TO DIFFERENT SUBSTRATES IN VITRO

Sylvia da C. F. Martins, Myrna C. Bonaldo² and Maurilio J. Soares.

Departamento de Ultra-estrutura e Biologia Celular and ²Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz / FIOCRUZ. 21045-900 Rio de Janeiro, RJ, Brazil.

Adhesion of epimastigote forms of T. cruzi to the cuticular epithelium of the rectal ampullae of the invertebrate host is a fundamental step in the process of differentiation into metacyclic trypomastigotes. This phenomenon can be reproduced in vitro when the parasites are incubated in a simple axenic culture medium (TAU3AAG).

In this work, aimed to characterize the best substrate for metacyclogenesis in vitro, we have compared the adhesion of T. cruzi epimastigotes to chitin, chitin analogous molecules and artificial substrates (glass coverslips coated with different substances). Stability of the adhesion in each one of these conditions was evaluated during processing for scanning electron microscopy (SEM). Parasites grown for 5 days in LIT medium were incubated for 1 hour in liquid TAU medium, in order to induce metacyclogenesis. Thereafter, the parasites were inoculated into TAU3AAG medium (10⁶ cells/ml) and then this medium was transferred to multiwell plates containing chitin, desmineralized chitin, chitosan flakes or sterile coverslips. The coverslips were either uncoated (control) or coated with glycol-chitosan, poly-L-lysine, bovine serum albumine (BSA) or poly-L-glutamic acid. The cells were grown for 24 hours and then the substrates were processed for SEM. The number of adhered parasites was evaluated in an area equivalent to 7,900 mm² in each of the above cited conditions.Our results show that although living parasites were able to attach to chitin in vitro, a total loss of adhesion occurred still during the initial stages of processing for SEM. On the other hand, a large number of epimastigote forms remained bound to chitosan and to glycol chitosan. The stability of adhesion to poly-L-lysine-coated coverslips (positively charged) was superior to that observed in the control, while few parasites remained adhered to the BSA- and poly-L-glutamic acid-coated coverslips (negatively charged). These results reinforce the hypothesis that the surface charge is one of the modulators during the initial stages of adhesion of T. cruzi epimastigote forms to the substrate. Further studies are being carried out to investigate the influence of adhesion to these substrates in the differentiation process.

This work has been supported by Capes, CNPq, PADCT/CNPq and FIOCRUZ.

FURTHER STUDIES ON HYDROPHOBIC ATTACHMENT OF TRYPANOSOMA CRUZI EPIMASTIGOTES

Schmidt, J.; Kleffmann T.; Kollien, A.; Schaub, G.A.

Department of Special Zoology, Ruhr University, 44780 Bochum, Germany

Trypanosoma cruzi multiplies and develops in the intestine of the triatomine vectors mainly as epimastigotes attached to the rectum cuticle. Previous work in this laboratory revealed that the rectum cuticle of Triatoma infestans has the same basic architecture as the external cuticle of arthropods. Notably, the luminal surface is covered by a waxy superficial layer. Chitin, by contrast, is a constituent of the procuticle and is not available at the luminal surface for binding of the flagellates. T. cruzi epimastigotes only attach by contact with the flagellum to the waxy superficial layer of the rectum cuticle, whereas in the midgut the flagellates remain free swimming and neither attach to the extracellular membrane layers nor to the microvilli. In vitro investigations of parasite binding on hydrophobic compared with hydrophilic substrates demonstrate that epimastigotes attach via hydrophobic interactions of the flagellum with the surface. An about 3 mm long area on the flagellum tip is specialized for the binding to the substrate. Small droplets, about 3-5 mm in diameter, of emulsified hexadecane bound selectively to this area. The attachment with the terminus of the flagellum ensures the settlement in a suitable region of the intestine and, in addition, the undulating movements of the resident trypanosomes may stir the external fluid to facilitate uptake of nutrients. While epimastigotes attach to all tested hydrophobic surfaces, vital trypomastigotes were never found attached to any of the substrates. This different behavior enables a separation of trypomastigotes from epimastigotes by hydrophobic chromatography using columns with octacosane-coated glass beads. If mixed populations with about 10% trypomastigotes are applied to the column, the „flow-through„ or „non-binding" peak fractions contain 90-95% trypomastigotes and intermediate forms, with recovery rates of more than 60%. In contrast to separation by ion exchange chromatography with DEAE cellulose, the new technique employs the natural binding mechanism and does not affect the surface charge of the trypomastigotes.

022

ADHESION OF LEPTOMONAS CYSTS TO ITS PARENTAL CELL.

Romeiro, A.; Attias, M. & De Souza, W.

Instituto de Biofísica Carlos Chagas Filho, UFRJ, Bl C, 21949-900, RJ, Brasil.

Leptomonas and Blastocrithidia are the only genera of trypanosomatids which produce cysts during its life cycle. These cysts were named by McGhee & Hanson (1962) as straphanger cysts. Cyst formation occurs by unequal division of mother-cell. Up to 4 cysts are produced and are kept attached to the parental individual from which they further detach. How straphanger cysts get adhered to parental promastigote is still unclear. Cells from Leptomonas sp. isolated from Oncopeltus fasciatus (Hemiptera: Lygaeidae) were observed by transmission electron microscopy and showed the pre-cystic individuals are attached to the mother cell by desmosome like structures. Those attachment sites occur between the flagellar membrane of parental and daughter cells. At the contact region the membranes of the participating cells are dilatated with a dense fibrilar region between them. In the intracellular side of the membrane a dense fibrous material is seen. Apparently it originates from the paraxial rod adjacent to it. These structures have not been described either for Blastocrithidia or Leptomonas, but probably existing all straphanger cysts producing trypanosomatids.

Sponsored by CNPq, PRONEX and FINEP.

023

SIALIC ACID RESIDUES OF HOST CELL ARE INVOLVED IN THE PROCESS OF TOXOPLASMA GONDII PENETRATION.

¹, Monteiro, V.G. & ^1,2 De Souza, W.

1-Lab. de Biologia Celular e Tecidual ,CBB, Universidade Estadual do Norte Fluminense (UENF), Campos RJ. 2-Lab. de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho (UFRJ), Rio de Janeiro, RJ.

Studies carried out with Toxoplasma gondii, the agent of toxoplasmosis, show significant quantitative differences in its capacity to penetrate into CHO cells (chinese hamster ovary) that expose different surface carbohydrates. CHO cells were used to evaluate the involvement of carbohydrates on the mechanism of parasite-host cell recognition. Five lineages of CHO cell were utilized: a parental lineage and four glycosylation mutants. Vero, LLCMK2 and CHO cells were cultured in medium supplemented with 5% fetal bovine serum (SFB). Cells were plated and interactions with tachyzoites of Toxoplasma gondii, RH strain, were performed using a parasite:cell ratio of 10:1. After the interaction, the cells were washed in PBS, fixed in Bouin fixative solution and stained with Giemsa. The W5 (CHO), Vero and LLCMK2 cells were desialylated by neuraminidase from Vibrio cholerae, for 1 hour and then incubated for 1 hour with the parasites and stained with Giemsa. Lec2 cells were sialylated with fetuin and Trypanosoma cruzi trans-sialidase for 30 minutes. Then the cells were washed in PBS and allowed to interact with the parasites for 1 hour. The control was done with cells incubated in MEM a medium without serum. The Lec2 cells were also incubated with the parasites in medium alpha-MEM, without serum, for 1 hour in presence of D-galactose. All experiments were submitted to statistical analyses using the t Student's test (p < 0.05). Our results demonstrate that all CHO cell lines could be infected and that the interiorization of parasites was higher in cells presenting sialic acid residues on the surface. Interiorization was partially inhibited by previous incubation of the cells with neuraminidase. On the other hand, Lec2 cells allowed to interact with parasites in the presence of D-galactose reduced in a larger extent their ability to ingest the parasites. Taken together these observations suggest that sialic acid and galactose residues exposed on the host cell surface are involved in the process of interaction with T.gondii.

Suported by: PRONEX, FINEP, FENORTE and CNPq.

024

ORIGIN OF THE PARASITOPHOROUS VACUOLE MEMBRANE IN VERO CELLS DURING TOXOPLASMA GONDII INVASION

Pacheco-Soares, C.¹ & De Souza, W.^1,2

¹ Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia- Universidade Estadual do Norte Fluminense- & ² Laboratório Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho - Universidade Federal do Rio de Janeiro.

Toxoplasma gondii is an important opportunistic pathogen causing congenital infections and severe complications in immunocompromised individuals. The intracellular tachyzoites reside within a vacuole which is incapable of acidifying or fusion with any membrane-bound organelle belonging to the host cell endocytic system. The composition of lipids and proteins in the parasitophorous vacuole (PV) is unknown, but lipids were likely to influence the ability of this compartment to interact with other vesicles within the host cell. To study the participation of the host cell plasma membrane in the formation of the PVM during Toxoplasma invasion, the surface of Vero cells was labeled with fluorescent probes - PKH26, DTAF and fluorescein-5-thiosemicarbazide - for lipids, proteins and sialic acid respectively. Vero cells were cultivated in round cover slips for 24 hours at 37^o in a 5% CO₂ atmosphere and labelled with 1- PKH26 (10mM) in glucose isotonic solution for 30 seconds at 4ºC; 0,5 mg/ml 2- DTAF{5-(4,6-diclorotriazinyl)aminofluorescein}, for 30 and 3- fluorescein-5-thiosemicarbazide (0,66 mg/ml) at a concentration of 0,5mM for 30 minutes at 4ºC. Thereafter, the Vero cells were allowed to interact with Toxoplasma gondii (strain RH) 50:1 for 15 and 30 minutes, 1 and 24 hours at 37ºC in a 5% CO₂ atmosphere. The cells were washed twice with PBS to remove extracellular parasites and fixed in 4% paraformaldehyde in 0,1M phosphate buffer (pH 7.2). The labeling of the parasitophorous vacuole membrane with these probes during Toxoplasma invasion was observed by confocal-laser scanning microscope. The results demonstrated that: 1- labeling with PKH26 the PVM showed a light staining while intravacuolar parasites were intensely labeled. After 24 hours of the incubation the intravacuolar parasites present the membrane labeled ; 2- with DTAF, 15 minutes the PVM was slightly labeled but the surface of intravacuolar parasites was intensely labeled. After 24 hours of interaction the intravacuolar parasites were not labeled and a disperse labeling of the host cell cytoplasm was observed. 3- Labeling with fluorescein-5-thiosemicarbazide was observed in surrounding PVM and some of the intravacuolar parasites. After 24 hours of incubation host cell and the intravacuolar parasites were not labeled. These observations show the participation of the host cell plasma membrane lipids and glycoproteins in the initial formation of the PVM, and that the plasma membrane proteins of the host cell are later on removed from the PVM. However the lipids remain in the PVM and after 24 hours are partially transferred to the intravacuolar parasites.

Supported by PRONEX, FAPERJ, FENORTE, CNPq and FINEP

025

TOXOPLASMA GONDII: MECHANISMS OF ENTRY INTO SKELETAL MUSCLE CELLS

Andrade, E.F*., Stumbo, A.C.**, Carvalho, L.** and Barbosa, H.S.*

*Dept. Ultra-estrutura e Biol. Celular, Instituto Oswaldo Cruz, FIOCRUZ, 21045-900, Rio de Janeiro, Brazil; ** Dept. Histologia e Embriologia, IB, UERJ, Rio de Janeiro, Brazil.

Toxoplasmosis is an widespread disease in human beings and many other warm-blooded animals, it is the most common oportunistic protozoan infection of the central nervous system and the skeletal fibers of individuals with AIDS. In this study we analyzed the biological aspects of the interaction of T. gondii with skeletal muscle cells (SMC), in vitro.

SMC were obtained from thigh muscles of 18-days-old mouse embryos. The tissues were dissociated with trypsin and versene in PBS. The isolated cells was suspended in Dulbecco's modified Eagle medium supplemented with horse serum, fetal calf serum, chick embryo extract, L-glutamine and antibiotics and then incubated at 37ºC in a 5% CO₂ atmosphere. The culture was maintained for 5-6 days to obtain the muscle fibers. Tachyzoites of T. gondii, RH strain, were used for interaction with host cells. After 1h of parasite-host cell contact the cells were fixed in 2.5% glutaraldehyde in 0.1M cacodylate buffer, pH 7.2, post-fixed in 1% O_sO₄ in the same buffer, dehydrated, critical point dried and coated with gold. The samples were observed in a Zeiss DSM 940 scanning electron microscope (SEM).

Entry of T. gondii tachyzoites into SMC during the adhesion stage was predominantly by the anterior region, where the conoid is located. By SEM some parasites appeared to penetrate with a counter-clockwise body torsion, suggesting that the invasion was an active penetration process. Parasites were observed attached to SMC by either the posterior end or by their sides. These parasites were also seen enveloped by host cell membrane expansions indicating that the uptake of parasites by endocytosis might also occur. Our observations also indicate a tendency of attachment of the parasite to the edges of the host cells.

There is a well-known controversy about the invasion process of the mammalian host cells by T. gondii. It is discussed whether the parasites are phagocytized or enter the cells by active invasion. Our observations are consistent with both processes, which appear to occur simultaneously in the skeletal muscle cells model.

Supported by CNPq, UERJ and FIOCRUZ.

026

PROTEIN PHOSPHORYLATION ON THE INTERACTION PROCESS OF MACROPHAGES BY TACHYZOITE FORMS T. GONDII.

Ferreira S.R¹., Cunha e Silva, N.L². Vieira, M.C.F.² & De Souza, W.^1&2

¹ Lab. Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, ²Laboratório de Ultraestrutura Celular Hertha Meyer,Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro.

Toxoplasma gondii is a protozoan parasite able of infecting most warm-blooded animal. This widespread infection (Toxoplasmosis) rarely gives rise to clinical disease in the adult, but causes severe illness and sometimes stillbirth in congenital infection in babies. The parasite has also gained more attention recently as the most common cause of focal central nervous system infections in patients with AIDS. We demonstrated in a previous study that treatment of macrophages with genistein, a tyrosine kinase inhibitor, or staurosporine, that inhibits a wide range of kinases, but specially kinase C, interferes in the process of adhesion and interiorization of T. gondii by macrophages. In the present study we attempted to detect phosphorylated proteins in macrophages at the onset of uptake T. gondii tachyzoites by macrophages. Macrophages were allowed to interact with tachyzoites and prepared for immunofluorescence and for immunoblotting assays. We used mouse monoclonal antibodies against phosphotyrosine, phosphoserine and phosphothreonine to probe phosphorylated residues on macrophages alone, parasites alone and interaction samples. Phallodin-rhodamine was used in the immunofluorescence experiments for observation of macrophage actin filaments behavior during the interiorization process. The attachment of tachyzoite forms to the macrophage surface triggered threonine phosphorylation and actin polymerization in the macrophage-parasite contact region. During the interiorization process were observed both phosphotyrosine and phosphoserine phosphorylation within parasithophorous vacuole. Non-infected macrophages of these same preparations showed positive fluorescence phosphotyrosine. Immunoblotting assays of the interaction detected tyrosine phosphorylated protein bands of 199, 85 , 52, 49, 47, 43 and 17 kDa. When macrophages were incubated with supernatant of tachyzoites culture the same phosphorylation pattern was detected on immunoblotting assays.

Supported by CNPq, FAPERJ, FINEP and PRONEX.

027

ABSENCE OF PHAGOLYSOSOMAL FUSION DURING THE INTERACTION OF TOXOPLASMA GONDII WITH SKELETAL MUSCLE CELLS in vitro

Andrade, E.F.*, Stumbo, A.C., **, Carvalho, L.** & Barbosa, H.S.*

*Depto. Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, FIOCRUZ, 21045-900, Brazil and ** Dept. Histologia e Embriologia, IB, UERJ, Rio de Janeiro, Brazil.

Toxoplasma gondii, a coccidian protozoan agent of toxoplasmosis, is able to infect many eukaryotic cells. In pacients with HIV infection, T. gondii is one of the most common opportunistic pathogens causing toxoplasmic encephalitis, and cysts of parasite has been found in skeletal muscle tissue. This finding is generally due to reactivation of a latent infection and is associated with intracellular multiplication of the parasite. The biology of T. gondii in host cells has been studied mainly with macrophages and line cells, and our purpose is to investigate the invasion by this parasite of primary culture of skeletal muscle cells (SMC).

SMC were obtained from thigh muscles of 18-days-old mouse embryos. The tissue was dissociated in 0.05% trypsin and in 0.01% versene in PBS. The cells were resuspended in DME medium supplemented with 10% horse serum, 5% fetal calf serum, 2% chick embryo extract, 1mM L-glutamine and antibiotics, and then incubated at 37ºC at 5% CO₂ atmosphere. The culture was maintained for 5-6 days to obtain the muscle fibers. For this study, the lysosomes of SMC were labeled with BSA-gold complex through 2 sequential incubations: 30 min/4ºC and 180 min/37ºC. The cultures were rinsed and then infected with T. gondii (tachyzoites, RH strain) for different periods of time (15 min to 5h). The cells were fixed for 1h at 4ºC in 2% glutaraldehyde, 4% paraformaldehyde in 0.1M cacodylate buffer containing 3.5% sucrose and 5mM CaCl₂, (pH 7.2), post-fixed in 1% O_sO₄ in the same buffer and processed as routine for transmission electron microscopy.

Our preliminary results demonstrate that after incubation the SMC with BSA-gold particles, the label was found in cytoplasmic vesicles. Even after 5h of infection, we could not detect BSA-gold particles in any parasitophorous vacuoles which indicates absence of phagolysosomal fusion in SMC during the interaction with T. gondii. This study is in accordance with reports using other host cells, in which phagolysosomal fusion does not occurs with live T.gondii, being this process observed only when fixed or degenerating parasites are used.

Supported by CNPq, UERJ and FIOCRUZ.

028

INTERACTION OF TRICHOMONAS VAGINALIS WITH McCOY CELLS

Alvarenga, V. L. de S., Mallavolta, V. A. A., Perone, D. & Cicarelli, R. M. B.

Faculdade de Ciências Farmacêuticas-UNESP, Universidade Estadual Paulista, Caixa Postal 502, Araraquara, 14801-902, SP, Brasil.

Trichomonas vaginalis is a well known parasitic protozoa of the human urogenital tract that causes sexually transmitted disease (STD). It is estimated that there are about ten million of new cases of trichomoniasis each year in the world. However, how the infection is established is not well elucidated. In order to better understand some mechanisms involved in the development of the disease, we have maintained axenically different strains of the parasite T. vaginalis isolated from infected women; these strains were maintained in Diamond medium at 35ºC by serial passages every three days and the strains were used for interactions with McCoy cells that were maintained in monolayer culture in Eagle medium. First of all, the parasites did not grow up in the Eagle medium without cells, but when inoculated the McCoy cells with 50 to 110 parasites, the cells died in 24 hours and many parasites were swimming free in the medium and exhibiting an amoebic shape, suggesting an activated-like state. The time for the cells death was dependent on the number of parasites inoculated reach the peak of parasitemia. Trying to elucidate if the parasites would produce any protease during the process, we have been electrophoresed in SDS-PAGE incorporated with gelatin the filtrated media from infected cells after the cell death and the peak of parasitemia, but no protease could be detected, even some proteases are detected in axenic culture by the same technique. These findings suggest that the parasite cytotoxicity would be mediated by other factors that are not proteases. Our studies are now undergoing to test another strains to verify if this effect would be strain-dependent and also characterize these factors.

Financial Support: NAC-LACAL/FCF-UNESP.

029

ROLE OF PUTRESCINE BIOSYNTHESIS ON THE TRITRICHOMONAS FOETUS: EFFECTS OF A PUTRESCINE ANALOGUE ON PARASITE METABOLISM, MORPHOLOGY AND ADHESION

Reis IA¹, López LB¹, Vannier-Santos MA², Martinez MP³, Yarlett N³, Costa-e-Silva F¹

¹ Laboratório de Biologia da Superfície Celular and ² Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; ³ Haskins Laboratories, Pace University, New York

The urogenital parasitic protozoan Tritrichomonas foetus produces large amounts of putrescine, a polyamine that plays a central role in several phenomena such as cell growth and differentiation. Trichomonad parasites produce additional ATP from carbamyl phosphate formed in the arginine dihydrolase pathway. The importance of this pathway to the parasite can be inferred from the significant amount of secreted putrescine in patients, suggesting a role in energy metabolism that may be important in a low carbohydrate environment, such as the vaginal cavity. The putrescine analogue 1,4-diamino-2-butanone (DAB), strongly inhibited polyamine biosynthesis in vitro. 20mM DAB remarkably impaired parasite proliferation after 16 h, nevertheless the overall motility was not affected. The DAB inhibitory effects were reversed by addition of putrescine to the culture medium. Ultrastructural observations revealed several alterations such as swollen endoplasmic reticulum cisternae. Enlargement of the hydrogenosome peripheral vesicles was associated with a general disorganization of the organelle matrix, ultimately resulting in its complete degradation. Trophozoites before and after DAB treatment were used for 240 h infection assays with HeLa epithelial cells. DAB treatment caused up to a 92% decrease in parasite adhesion to epithelial cells. Taken together these data indicate that polyamine biosynthesis is important in the metabolism, in vitro growth and cytophatic effects of T. foetus.

Supported by FINEP, CNPq, PRONEX, FAPERJ and CAPES.

030

CHARACTERIZATION OF AN INTRACELLULAR EPIMASTIGOTE-LIKE FORM OF TRYPANOSOMA CRUZI

Almeida-de-Faria, M., Haapalainen, E. F.*, Colli, W. & Alves, M.J.M.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP 26077, 05599-970, São Paulo, Brazil *Centro de Microscopia Eletrônica, UNIFESP/EPM.

Trypanosoma cruzi has a complex life cycle with different developmental forms observed in the vertebrate and invertebrate hosts. These forms can be identified by light microscopy according to: (a) the overall morphology of the parasite; (b) the position of the kinetoplast relative to the nucleus; (c) the position of flagellum emergence. These criteria define three differentiation forms of T. cruzi: (1) amastigote, a non-flagellated intracellular multiplicative form in the mammalian cell; (2) trypomastigote, the flagellated infectious form to the vertebrate host, and (3) epimastigote, the flagellated multiplicative form in the invertebrate host. One unresolved question is whether epimastigotes may appear as obligatory intermediates in the mammalian intracellular transformation of amastigotes to trypomastigotes.

Following the intracellular cycle of T. cruzi, clone CL-14, in mammalian cells, the presence of an intermediate form in the 6th day of infection was observed. This form, which was denominated epimastigote-like, appeared in the differentiation of amastigote to trypomastigote and was morphologically very similar to the epimastigote form, as it appears in liquid axenic culture media.

A systematic study was undertaken using clone CL-14 of T. cruzi infecting CHO-K1 cells cultivated in RPMI medium. Cells were scrapped with a rubber policeman in the 6th day of infection and the parasites, separated from cell debris by centrifugation, were analysed by light microscopy. The folowing criteria established the similarity between the epimastigote-like forms and true epimastigotes obtained from liquid axenic media: (a) both displayed intense reaction by immunofluorescence using a bona fide monoclonal antibody specific for the epimastigote form; (b) both were susceptible to complement lysis; (c) both have on their surfaces the sugar residues a-D-mannose and a-D-glucose, [(GlcNAc)2NeuNAc], and [b-Gal(1-3)GalNAc] as recognized, respectively, by concanavalin A, wheat germ agglutinin and Arachis hypogea agglutinin; (d) both have negative charges on their surface. The results strongly support the notion that epimastigotes are intermediate forms in the intracellular amastigote-trypomastigote transformation and, thus, that epimastigotes are also present in human infections. Electron microscopy studies with both forms are presently under way.

M. Almeida-de-Faria is a doctoral student from CNPq.

Work supported by FAPESP and CNPq/PADCT.

CELL TRANSFORMATION IN HERPETOMONAS ROITMANI: FROM OPISTHOMORPHS TO PROMASTIGOTES

Palmié, I.¹, Faria-e-Silva P.M ^1,2, Cunha-e-Silva, N.¹ & de Souza W.^1,3

¹.Lab. Ultraestrutura Celular Hertha Meyer, IBCCF, UFRJ, Rio de Janeiro, RJ ².Depto. de Ciências Biológicas, EFOA, Alfenas, MG. ³.Lab. de Biologia Celular e Tecidual, CBB, UENF, Campos dos Goytacazes, RJ.

Recent work has suggested that Herpetomonas roitmani belongs to group C of Herpetomonas genus (Teixeira et al., J. Parasitol., 83(1): 58-65, 1997). This group of trypanosomatids displays interesting features including the presence of cells with a shape that resembles choanomastigotes of Crithidia species, but the kinetoplast is located posteriorly to the nucleus, like in opisthomastigotes of Herpetomonas species. The term "opisthomorphs" has been suggested then to identify these forms. As we have described before, H. roitmani can reach 98% of opisthomorphs in early cultures. Our recent results indicate that the process of cell transformation in H. roitmani may occur from opisthomorphs to promastigotes instead from pro- to opisthomastigotes as observed in other Herpetomonas species. The number of promastigotes of H. roitmani increases in some conditions including the composition of the medium, e.g. cultures adapted in proline medium show more promastigotes. Also, the presence of protein kinase inhibitors like staurosporine and genistein increases the percentage of promastigotes. Preliminary studies by SDS-PAGE analysis show that notable changes occur in the pattern of both total proteins and biotinylated surface proteins of H. roitmani related to the process of cell transformation.

Supported by CNPq, PRONEX, CAPES and FINEP

032

LEISHMANIA CHAGASI: METACYCLOGENESIS CHARACTERIZATION OF A NEW WORLD SPECIES.

da Silva, L.H.P.¹ & Saraiva, E.M.B.¹

1. Laboratório de Imunobiologia das Leishmanioses, Departamento de Imunologia, Instituto de Microbiologia - UFRJ.

Promastigote forms of Leishmania are capable of differrentiate from a non-infective (procyclic) to an infective stage (metacyclic) within the alimentary tract of their sandfly vector and in axenic medium. Metacyclic promastigotes are the only forms inoculated by the insect vector in the mammalian host.

In this study, we characterize the metacyclogenesis of L. chagasi during their growth in vitro, by complement lysis resistance, morphological characterization, lectin agglutination and macrophage survival.

L. chagasi was mantained at 27ºC in Schneider or brain heart infusion medium supplemented with 10% fetal calf serum and 2% human urine. Promastigotes taken from different time points of their growth culture were used for all the different assays. Complement lysis resistance assay was performed incubating promastigotes with a pool of fresh normal human sera serial diluted in PBS + Ca⁺⁺ and Mg⁺⁺ at 37ºC in 7% CO₂ for 45 minutes. Viable/motile promastigotes were scored in a haemocytometer, and the percentage of survival determined. In a serum dilution of 1/8, 19.5% of log phase promastigotes survival was obtained compared with a 87,5% survival of stationary phase promastigotes. Infectivity in vitro was tested using mouse resident peritoneal macrophages adhered to glass coverslips. Promastigotes opsonized by complement were added to the macrophages in a 1:10 ratio and incubated at 37ºC/ 7% CO₂. After 1 hour, free promastigotes were washed out, some coverslips were fixed and others were incubated for more 4 days in the same conditions above. For each culture over 600 cells were counted to determine the percentage of macrophages infected and the number of parasites per cells. Lectin agglutination of log and stationary phase promastigotes was evaluated with peanut lectin (PNA). Parasites were incubated with serial dilutions of PNA in PBS, at room temperature for 45 minutes, and the percentage of agglutination was determined. A morphological study of PNA (-) forms showed short, slender, highly active promastigotes with elongated flagella.

Our results showed a growth cycle dependent generation of an infective stage of L. chagasi. This stage showed increased complement resistance and infectiviy for macrophages, loss of PNA binding sites and a typical morphology of metacyclic promastigotes.

Supported by: WHO, Pronex, FUJB-UFRJ, CNPq.

033

PROTEIN PROFILE IN HERPETOMONAS SAMUELPESSOAI DURING CELL DIFFERENTIATION.

Santos, A^.,.L.S.; Angluster, J. and Soares, R.M.A^,.

Departamento de Microbiologia Geral, Instituto de Microbiologia Professor Paulo de Góes (IMPPG), Universidade Federal do Rio de Janeiro (UFRJ), 21944-970, Cidade Universitária, Rio de Janeiro, Brasil.

Parasite proteins are involved in host cell recognition and penetration. Surface proteins are important antigens which elicit humoral and cell-mediated responses. Herpetomonas samuelpessoai is a non-pathogenic trypanosomatid which can be easily cultivated in a chemically or complex medium (Roitman et al., 1972) which shares important common antigens with Trypanosoma cruzi, the agent of Chagas' disease (Souza et al., 1974). In the present work, total protein extracts of H. samuelpessoai cultivated in chemically defined medium for 48 hours at 28⁰C supplemented or not with 4% of dimethylsulfoxide (DMSO) was analyzed on 12% SDS-PAGE (Laemmli, 1970). The gels were silver stained. Protein profiles showing about 40 well defined bands ranging from 15 to 200 kDa were obtained in both systems. There were generally minor differences in protein profiles between H. samuelpessoai grown in the ausence (85% promastigote and 15% paramastigote) and presence (55% pro and 45% paramastigote) of DMSO with the marked exception of prominent 66, 45, 30, 20 and 15 kDa proteins which were preferentially expressed in control system. Further studies will determine if these proteins are related to the differentiation process.

Financial Support: CNPq, CEPG-UFRJ, FINEP.

034

LEISHMANIA (V) BRAZILIENSIS: LECTIN AGGLUTINATION, COMPLEMENT LYSIS SUSCEPTIBILITY AND IN VITRO INFECTIVITY STUDIES.

CAMURATE,M^@; COSTA,KA^@ & SARAIVA,EMB^@

@ Laboratório de Imunobiologia das Leishmanioses, Departamento de Imunologia, Instituto de Microbiologia - UFRJ

Leishmania are digenetic parasites that multiply as intracellular amastigotes in macrophages of their vertebrate host and as extracellular promastigotes in the midgut of their sandfly vectors. The life-cycle of Leishmania parasites within the sandfly includes the sequential development of promastigotes from a non-infective- procyclic form to an infective - metacyclic - stage. Procyclic and metacyclic promastigotes can also be obtained during parasite growth in axenic medium.

L. braziliensis promastigotes mantained at 27⁰C in Schneider's medium supplemented with 10% Fetal Calf Serum and 2% human urine was used for all the assays, at different time points of their growth. Complement lysis assay was performed by exposing promastigotes to serial dilutions of normal fresh human serum in PBS + Ca⁺⁺, Mg⁺⁺. Viable/motile promastigotes were scored in a haematocytometer. Infectivity in vitro was tested using mouse resident peritoneal macrophages adhered to glass coverslips and parasites at a 1:10 ratio. After a 1-hour at 37⁰C / 5%CO₂, free promastigotes were removed by repeated washings and cultures were part fixed and part incubated during 4 days at 37⁰C / 5%CO₂. For each of the triplicate culture, at least 600 cells were counted and the percentage of infected macrophage, the mean number of parasites/cell and the endocytic index determined. Lectins with specificity for D-mannose, D-galactose, L-fucose, N-acetylgalactosamine and N-acetylglucosamine were used in the agglutination assays.

Our preliminary results indicate that stationary phase promastigotes of L. braziliensis are more infective to mouse peritoneal macrophages, more resistant to lysis by human serum and less able to binding Canavalia ensiformis (Con A), Ricinnus communis II (RCA II) and Jacalin than log phase promastigotes.

Supported by : WHO, Pronex, FUJB-UFRJ, CNPq.

035

CELLULAR DIFFERENTIATION IN TRYPANOSOMATIDES ISOLATED FROM PLANTS: USEFULNESS OF MORPHOLOGICAL CHARACTERS TO IDENTIFY PHYTOMONAS

Sousa, M.A., Sá-Xavier, C.^* (* CNPq Fellowship) , Santos, S.M. & Branco, D.C.B.

In the present work we studied at the morphobiological levei eight isolates of plant trypanosomatids, which have been confinned as Phytomonas or Herpetomonas by at least two of the following approaches: isozyme analysis, reactivíty with selected monoclonal antibodies or lectins, arginase detection, analysis of restriction sites in some RDNA sequences, and by molecular hybridizations using probes derived from KDNA, RDNA or SL genes. The Herpetomonas were: H. davidi (previously named P. davidi) and the isolate of Euphorbia hyssopifolia (Attias & De Souza, 1986). The Phytomonas were: P. ser ,pens (Jankevicius et al. 1989), the isolates obtained from Euphorbia pínea (Doflet et al. 1982), Jathropha macrantha (Burstein 1981), Allwnanda cathartica (Kastelein & Parsadi 1984), Citrus bergamia (Conchon et al. 1989), as wefi as the so-called "H." mcgheei (Itow-Jankevicius et aí. 1993). Three well-characterized Herpetomonas species isolated from insects were also studied as referendes We were looking for a character only shared by the Phytomonas isolates, and which could be considerei of diagnostic value. Then, we followed the cellular differentiafion of these isolates in LIT medium at 27.3oC, at 24 hr intervals, from 48 to 120 hr. The cultures were started with 5xlO' cells/ml seeded in 4 nd-volumes of medium distributed in l6xl5Omm screwcap tubes. The percentage of the different evolutive stages was determinei by g about 300-500 randomly chosen cells in Giemsa-stained srnears.

Paratnastigotes and opisthomastigotes were detected only in isolates identified as Herpetomonas. On the other hand, in afi Phytomonas cultures occurred both a type of promastígote presenting a very short flagehum and sin-úlar forms without an apparent flagellum. These afiagellated forrns are very peculiar and were found at rates ranging (mean) from I. 9% ("H". mcgheei) to 51.4O/o (Phytomonas from Allamanda cathartica), but they were not seen in the Herpetomonas. They had already been seen in P. serpens, "H". mcgheei (in the plant) and other Phytomonas spp., but have not been reported in other genera. Then, we beheve they are useful morphological markers to identify Phytomonas. It is worthy mentioning that these aflagellate forras are distinct from arnastigotes of Leishmania and T@nosoma, as well as from "cysts" of Leptomonas. ln the majority of Phytomonas cultures we also observed a type of division producing one cell with flagellum and another without it; it seems possible that this division would be a source of aflagellated forms. Otherwise, although long twisted promastigotes are comtnon in Ph onas in the host plants, they were rare or not seen in the majority of the cultures

examined herein. Our data also evidenced that the presence of paramastigotes in trypanosomatids from plants should be considerei a clue, to be confinned, that the isolate can be a Herpetomonas sp.

036

OBSERVATIONS OF NUCLEAR BEHAVIOR DURING THE CLOSED MITOSIS OF TRITRICHOMONAS FOETUS

Ribeiro K. C. & Benchimol M.

Laboratorio de Biologia Celular e Tecidual, CBB, Universidade Estadual do Norte Fluminense, Av. Alberto Lamego, 2000 Campos, RJ

Contrary to what happens in higher eukaryotes that display nuclear envelope breakdown during mitosis, lower eukaryotes maintain intact the nuclear compartment. In the closed mitosis type of cell division, genome segregation must be accomplished under the constrains of the nucleus. Tritrichomonas foetus, a bovine parasite that represents one of the most distant branches on the eukaryotic phylogeny, was used in this study as a cell model to trace the genomic segregation pattern. Ethidium bromide, a DNA probe was incubated with mitotic cells to yield stained nuclei. Confocal laser scanning microscopy allowed a three dimensional approach of the DNA content based on serial optical sectioning. Differential interference contrast coupled to the fluorescent labeling of the nucleus rendered information about the axostyle participation on the nuclear constriction process. The overall shape modification of the nucleus could be followed on mitotic cells in different phases of division. Furthermore, our results demonstrate a circular arrangement of 6 chromosomes around a central axis. The chromosomes could be visualized as dense, fluorescent round masses. In conclusion, we postulate that during nuclear compartment partition, nuclear shape becomes elongated, then suffers a torsion movement produced by the two daughter cells and subsequently it is strangulated by the aid of the axostyles. Parallel to this, we suggest a reevaluation of the mitotic evolutions current scheme, defending the hypothesis that the closed mitosis present in the phylum Parabasalia reflects an intermediary evolution pattern of the opened type of mitosis present in higher eukaryotes. We can imagine how might have occurred the transfer of the genome from membrane systems to skeletal systems, such an event that occurred more than a billion years ago when cell compartmentalization proliferated.

Supported by: FENORTE, CNPq, FINEP, PRONEX

037

ADDITIONAL STUDIES ON THE RESERVOSOMES OF TRYPANOSOMA CRUZI

Porto-Carreiro,I., Moreira-Leite, F., de Souza, W. and Cunha-e-Silva, N.

Lab. Ultraestrutura Celular Hertha Meyer, Inst. Biofísica Carlos Chagas Filho, UFRJ, CCS, Bloco G, Rio de Janeiro, 21949-900, Brasil.

Reservosomes are storage organelles of Trypanosoma cruzi localized at the posterior end of epimastigote forms (Soares & De Souza, 1988, J. Submicrosc Cytol. Pathol., 20:349). These acidic compatments, which accumulate proteins and lipids internalized by endocytosis, were classified as late endosomes rich in cruzipain (Soares et al., 1992, J. Cell Sci., 102:157). In the present work, we compared the ultrastructural aspect and storing capacity of reservosomes of three different strains of T. cruzi. Reservosomes from Dm28 strain presented huge lipidic inclusions and were more irregular in shape than those from Y and CL-Brener strains. CL- Brener and Dm28 strains presented respectively the largest and the smallest storing capacity. Ammonium chloride or cloroquine treated parasites had their intracellular albumin traffic slowed down, which allowed us to observe many events of vesicle fusion. We also analized the twenty four-hour endocytosis of albumin: whereas the majority of reservosomes were full of albumin-gold particles, few of them were still lacking the protein. To begin studying the role of those unloaded reservosomes, we assayed simultaneous uptake and intracellular traffic of proteins internalized either by receptor mediated endocytosis (transferrin-gold complexes) or fluid phase endocytosis (non-conjugated horseradish peroxidase). The fluid phase marker reached reservosomes much quicker, but both ligands were found in the same reservosomes after 30 minutes of endocytosis. In these parasites we could still find unloaded reservosomes.

This work has been supported by PRONEX/MCT, CNPq, FINEP, FAPERJ and UFRJ.

038

THE PERIPHERAL VESICLES OF GIARDIA LAMBLIA

Lanfredi-Rangel, A. ¹, Attias, M.², Carvalho, T.U.^1,2 & de Souza, W.^1,2

¹Centro de Biociências e Biotecnologia, UENF, and ² Instituto de Biofísica Carlos Chagas Filho, UFRJ

Giardia lamblia is a primitive eukaryote that parasitizes the duodenum of most mammalian species, including man. Previous morphological studies have shown that this protozoan does not present organelles such as mitochondria and peroxisomes and that the elements of the endoplasmic reticulum and Golgi complex do not show the typical organization found in other eukaryotic cells. However, some special structures such as the adhesive disc, median body and a system of peripheral vesicles are observed in trophozoite forms of G. lamblia. In previous studies it has been shown that macromolecules ingested by trophozoites of G. lamblia concentrate in the peripheral vesicles and that most of the vesicles contain acid phosphatase, a lysosome marker. In the present study we analysed in more detail the participation of the peripheral vesicles in the endocytic system of the protozoan. Trophozoites (Portland-1 strain) were cultivated, washed and then incubated in the presence of Lucifer yellow and observed in a Confocal Laser Scanning Microscope. A strong labeling of the peripheral vesicles was observed even after short incubation times (15 minutes), persisting after longer incubation times (120 minutes). The cells were also incubated for periods varying from 5 to 120 minutes in the presence of horseradish peroxidase, then washed, fixed in glutaraldehyde, incubated in a diaminobenzidine containing medium for localization of peroxidase activity, post-fixed with osmium tetroxide and processed for transmission electron microsocope using standard procedures. Peroxidase activity was always observed in the peripheral vesicles, independent from the incubation time. These observations, in association with those reported previously, suggest that the peripheral vesicles of trophozoites of G. lamblia represent, at the same time, early endosomes, late endosomes and lysosomes. Incubation of trophozoites in a cytochemical medium designed for the detection of glucose-6-phosphatase, a marker of the endoplasmic reticulum, showed intense labeling of the outer nuclear membrane, in profiles of the endoplasmic reticulum, some of which seem to establish contact with the peripheral vesicles, and within some of the peripheral vesicles. Three-dimensional reconstruction of serial thin sections and electron tomography of thick sections added new information to inter-relationships between the endoplasmic reticulum and the peripheral vesicles.

Supported by PRONEX, FINEP, FAPERJ, FENORTE and CNPq

039

UNUSUAL HYDROGENOSOMES

Miranda, K. R.¹ & Benchimol, M.²

¹Lab. de Biologia Celular e Tecidual, CBB, UENF, Campos-RJ ²Universidade Santa Ursula, Rio de Janeiro-RJ

Hydrogenosome is an organelle that was initially found in protozoa of the Trichomonadida order and which contains enzymes that participate in the metabolism of pyruvate formed during glycolysis and was the site of formation of molecular hydrogen. Later on this organelle was described in some fungi, in a number of anaerobic rumen ciliates and in a certain free living ciliates. The hydrogenosomes were described as spherical or slightly elongated granules with 0.5-2.0mm diameter, closely associated to cytoskeletal structures such as the axostyle and costa (paraxostylar and paracostal granules). We have shown previously that the hydrogenosome was enveloped by two closely apposed unit membranes. A flattened membrane-bounded vesicle was found at the periphery of the hydrogenosome. This compartment accumulates high levels of calcium, showing that these organelles are involved in the regulation of intracelular calcium content. This organelle is formed by division of preexisting ones and occasionally connections of hydrogenosomal membranes with endoplasmic reticulum can be seen. The aim of this work is to re-analyse the shape of the hydrogenosome of Monocercomonas sp. In these cells we detected an unusual elongated snake-like hydrogenossome. Serial sectioning showed that these forms are a common characteristic of this species and do not correspond only to the hydrogenosome step division. On the other hand, when trichomonad cells were treated with metronidazol compounds, elongated hydrogenosomes were easily seen. These present abnormal shape and size, with internal membranous structures resembling mitochondrial cristae. The same mechanism of cristae formation is also observed when these cells are activated during endocytosis. In other circunstances such as when cells are under stress, pleomorphic hydrogenossomes were found. We discuss the possible reasons for these findings.

Supported by: CNPq, FINEP, PRONEX and AUSU

040

DIFFERENT NUCLEAR ORGANIZATION DURING TRYPANOSOMA CRUZI LIFE CYCLE

Porto, R.M., Elias, M.C.Q.B., Haapalainen, E., Mortara, R.A. and Schenkman, S.

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina Universidade Federal de São Paulo, R. Botucatu 862 /8^o, 04023-062 São Paulo, S.P. Brasil.

Trypanosoma cruzi life cycle includes proliferative forms (epimastigotes and amastigotes) and infective trypomastigote forms, which do not divide. We found that during differentiation from one form to another there are dramatic changes in nuclei morphology and organization. In dividing forms, the nucleus is round, contains a large and central nucleolus and abundant euchromatin as seen by optical and electron microscopy. In contrast, the nucleus of infective trypomastigotes is elongated, with abundant heterochromatin material and no apparent nucleolus. A monoclonal antibody (mAb 3F4), that recognizes an antigen associated with the chromatin, strongly stained nuclei of the proliferative but not of infective forms. When trypomastigotes were solubilized in detergent, the nucleus increased in size, exposing the epitope for mAb 3F4. The epitope was inaccessible in nuclei purified from trypomastigotes in absence of detergent, unless when mechanically disrupted. The antigen recognized by mAb 3F4 could be removed from the nucleus by extraction with 1M NaCl, or by DNase treatment, but not by treatment with RNase. Addition of the salt extracts to the nuclear remnants reconstituted the mAb 3F4 epitope. Therefore 3F4 antigen may be associated with the core histones and revealed different chromatin organization and accessibility in proliferative versus infective stages. These differences might be related to the differential gene expression, found in the various parasite developmental stages.

Financial support: CNPq, CAPES, PADCT, FAPESP.

WHAT TYPES OF HAEMOCYTES ARE RESPONSIBLE FOR THE CONSTITUTION OF HAEMOCYTIC CAPSULES IN PANSTRONGYLUS MEGISTUS?

Cipullo, R.*; Barraco, M.**; Gonçalves, E.K.*; Scaff, R.M.C.*

*Departamento de Ciências Morfológicas. **Departamento de Biologia, Embriologia e Genética.

Centro de Ciências Biológicas da Universidade Federal de Santa Catarina. Caixa Postal 476, Florianópolis, 88010-970, SC, Brasil.

Haemocytic capsules development is one of the defense process used by the haemocytes of insects. It occurs when large structures that cannot be fagocitesed invade or are introduced in the haemocele, and consists in the adhesion of haemocytes around non-self material, constituting one capsule whose function is to isolate these materials from the normal tissues. In Panstrongylus megistus (Hemiptera Reduviidae) we have observed haemocytic capsules development around pollen grain of Hibiscus sp, and we have described his own ultrastructure. Nimphs of P. megistus of the fifth instar where inoculated in the haemocele with Hibiscus pollen grains, being dissecated 3 hours, 48 hours and 4 days after treatment. The capsules finded in the haemocele were treated by the sequential routine process for electron-microscopy. These capsules resemble the humoral or humoral-celular types, and in the present work we analyse mainly what types of haemocytes would be involved in the encapsulation process. The haemocytes of Panstrongylus megistus are of the following types: prohemocytes, plasmatocytes, granulocytes, coagulocytes, oenocytoids and adipohemocytes. Among insects its relativally well stablished that the main haemocytes involved in capsules development are the granulocytes and the plasmatocytes. In P. megistus the granulocytes have a well develloped rough endoplasmic reticulum wich shows various degrees of cisternae dilatation, a large amount of free ribosomes and a central nucleus with scattered chromatin masses; the plasmatocytes shows similar characteristics, and, besides, lysosomes and a large vacuolation. Considering that the capsules of P. megistus around pollen grains are constituted of two non clearly delimiting regions, one proximal to the pollen, with a necrotic appearence, and one distal constituted with a "mass" of cells remmants and some complete cells, but with many alterations, the exact recognition of the cells involved in capsule's development is difficult. Nevertheless, among the cellular debris we can find many structures and characteristics typical of granulocytes and plasmatocytes, like granules, lysosomes, large vacuolation, fragments of rough endoplasmic reticulum, and nuclei wich resembles these of that cells. The presence of cytolysomes and multivesicular bodies, structures related to lysosomes action, have to be take into acount, too, because of the presence of these organelles in plasmatocytes. Moreover, some integral capsule's cells, although with many alteractions, shows some characteristics that resembles granulocytes and plasmatocytes. These data suggests that also in P. megistus granulocytes and plasmatocytes are the main haemocytes involved in capsule's development, and we discuss about this.

042

THE CYTOSTOME OF TRYPANOSOMA CRUZI EPIMASTIGOTES IS A CYTOSKELETAL STRUCTURE ASSOCIATED TO THE FLAGELLAR COMPLEX.

Kendi Okuda^@, Mónica Esteva^#, Elsa L. Segura^#, A. Tania Bijovsky^@

^@Depto. Parasitologia, ICB-USP, 05508-900, São Paulo, Brasil. ^#INDIECH, Bs. As. Argentina.

Proliferative forms of T. cruzi, amastigotes and epimastigotes, have a specialized structure, the cytostome, formed by an invagination of the flagellar pocket's membrane which is surrounded by microtubules and often accompanied by a row of vesicles. All this assemblage penetrates deeply into the cytoplasm frequently overpassing the nucleus. This structure is described as an important organelle for the parasite's endocytosis.

In previous reports we demonstrated that the monoclonal antibody (mab) 2C4, made-up against the isolated flagellar complex of T. cruzi epimastigotes, recognizes a polypeptide with molecular weight around 85 kDa, both in detergent soluble and insoluble fractions of T. cruzi epimastigotes as well as in isolated flagella of these forms.

Immunofluorescence assays detect the antigen as a small spot at the flagellar pocket region. The mab does not detect any structure in trypomastigote or amastigote forms of T. cruzi, neither in other trypanosomatid epimastigote forms, e.g., T. conorhini and Blastochritidia culicis.

Immunogold labeling of ultrathin sections of epimastigote forms reveals gold particles at the opening of the flagellar pocket, concentrated on the cytostome region. Immunocytochemistry of epimastigote whole-mount cytoskeletons reveals the labeling on an array of 3-4 microtubules that is attached to the flagellum and appearing to run towards the nuclear region. Ultrastructural observations of negatively stained isolated flagella show, in their posterior region, at the level of the flagellar pocket, the presence of a microtubular structure that constitutes the cytoskeleton of the cytostome.

Immunolocalization of a specific protein of T. cruzi epimastigotes cytostome allowed to establish, for the first time, the physical relationship between an endocytic organelle and the flagellum.

Supported by FAPESP and CNPq. K.O. is a fellow of FAPESP

043

PARTIAL CHARACTERIZATION OF ADHESIN FROM TRITRICHOMONAS FOETUS

Silva, N.S.^*, Dias Filho, B.P.⁺ & De Souza, W. ^*

^*Laboratório de Biologia Celular e Tecidual and ⁺Laboratório de Fisiologia e Bioquímica de Microrganismos - Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense (UENF), Campos - RJ, Brasil.

When Tritrichomonas foetus was lysed by sonication, followed by low-speed centrifugation and further fractionated by ultracentrifugation, resulted in a 100,000g pellet with hemagglutining activity. Although the resulting supernatant failed to mediate parasite-erythrocyte interaction its presence in the incubation medium, completely inhibited attachment of the parasites to the erythrocytes. The treatment of adhesin with Trypsin, and addition of sugars in the interaction medium (D(+) galactose, a-lactose and D(+) galactosamine) inhibit the hemagglutination, suggesting the protein composition of this adhesin and possibly acting as a lectin-like adhesin. 20ml of a erythrocyte ghost suspension were added to 20ml of a biotinylated 100,000g pellet suspension, applied to SDS-PAGE on 10% gel, transfered to nitrocellulose, and revealed with Avidin-PA. A single protein band of 100kDa was present. This 100kDa band was not observed in gels of erythrocytes or the 100,000g pellet alone. Male CF-1 mice were immunized with 20mg of the 100kDa protein to obtain an antibody against the adhesin. For immunofluorescence microscopy all incubations were carried out at 4^oC. Living parasites were initially adhered to poly-L-lysine coated coverslips, incubated with the anti-adhesin antibody (1:100) for 30 minutes followed by FITC-labelled anti-mouse IgG (1:100) for 30 minutes. The specimen was then fixed in 4% paraformaldehyde, 0,1% glutaraldehyde in PHEM buffer (pH 7,2), for 15 minutes at room temperature. The preparations were analysed by confocal-laser scanning microscopy. Immunofluorescent analysis showed a ponctual label of adhesin on the surface of the parasite. For electron microscopy, T. foetus was washed in PBS, fixed in 4% paraformaldehyde, 0,1% glutaraldehyde in 0,1M cacodylate buffer (pH 7,2), for two hours at room temperature. Cells were infiltrated with cryoprotector (PVP + sucrose 2.3M) and frozen. Cryo-sections were obtained and incubated with the first antibody [against adhesin 100kDa (1:100)] for 3 hours followed by incubation with the second antibody [anti-mouse IgG (1:100)] conjugated to coloidal gold particles (10nm). The material was then infiltrated in PVA and observed in a Zeiss 900 Transmission Electron Microscope. Observations by electron microscopy confirmed the results observed by fluorescent microscopy. Trichomonas galinae did not express hemagglutinating activity nor surface fluorescence when incubated with anti-adhesin antibody, confirming the specificity of the anti-adhesin antibody to T. foetus.

Supported by PRONEX, FAPERJ, FENORTE, CNPq and FINEP

044

IMMUNOLOCALIZATION OF TWO DIFFERENT MEGADALTON PROTEINS DETECTED IN THE TRYPANOSOMATID CRITHIDIA LUCILIAE THERMOPHILA.

Moraes, N; Milder, R.; Baqui, M. M. A.; Pudles, J.

Instituto de Ciências Biomédicas II, Universidade de São Paulo, São Paulo, 05508-900, SP, Brasil.

We have previously shown by biochemical studies, the presence of two different megadalton proteins in a trypanosomatid. Crithidia luciliae thermophila presents a giant insoluble protein associated with the highly stable cytoskeleton (Ci 1300) (Mr @ 1300 k) and a detergent soluble megadalton protein (Cs 2300 - Mr @ 2300 k) which is a peripheral component of plasmatic membrane. Specific antiserum were raised against these giant proteins, as revealed by immunoblotting and immunoprecipitation of the proteins metabolically labeled with [35S]methione.

In this work we demonstrate the cellular localization of these two different proteins in immunofluorescence assays and immunogold electron microscopy of thin sections and whole cytoskeleton preparation.

Immunofluorescence localization of soluble Cs 2300 performed on fixed parasites showed a punctate fluorescence at the anterior end of cell body. Similar localization was observed with Ci 1300 on cytoskeleton preparation. Immunogold labeling of Cs 2300 showed a reaction near the flagellar pocket and at the beginning of the flagellum. Ci 1300 was also detected at the anterior region of cell without a precise localization.

In contrast with other trypanosomatids, we have detected for the first time, two different megadalton proteins in C. l. thermophila, a detergent soluble protein and a insoluble protein, both localized at the anterior end of the parasite.

045

LOCALIZATION OF SPOROZOITE SURFACE PROTEIN 2 (SSP2) AND CIRCUMSPOROZOITE LIKE PROTEIN (CS-LIKE) IN BLOOD-STAGE OF PLASMODIUM YOELLI BY IMMUNOELECTRON MICROSCOPY.

Porrozzi, R; Soares, R.; Meuser, M. and Meirelles, M.N.L

Laboratório de Ultra-estrutura Celular, Instituto Oswaldo Cruz.,FIOCRUZ, 21045-900, RJ, Brasil.

The complex life cycle of malaria parasites is characterized by infection of both invertebrate and vertebrate hosts where morphological changes are frequently followed by stage specific antigen expression. However, some of those antigens can be expressed in two or more developmental stage. PySSP2, a140-kDa sporozoite protein, is present in the micronemes of sporozoites, on sporozoite surface, and throughout the liver stage (M.Aikawa et al. 1990.Bull. W.H.O. 68: 165). The Plasmodium falciparum homologue, PfSSP2 has been shown to be the previously described 90-kDa thrombospondin-related anonymous protein (TRAP) and is present on sporozoite surface, micronemes , for the first 4 days of the liver stage and at low levels by erythrocytic stage of some strains. CS protein is the major surface protein of sporozoites but is also found in micronemes. This protein is brought into hepatocytes when sporozoites invade those cells and a CS-like protein has been demonstrated in invasive blood stage of malaria parasites (A.H.Cochane et al. 1990. Bull. W.H.O. 68: 181).

Immunoelectron microscopy was performed on red blood cells from mice infected with 17X (NL) strain of P. yoelli . Infected erythrocytes were fixed for 30 minutes at 4ºC in 4% paraformaldehyde, 0,2 % glutaraldehyde in 0.1M cacodilate buffer, pH 7.4 and were embedded at low temperatures in Lowicryl resin. Sections were cut with a diamond knife, mounted on nickel grids and labeled with Mabs NYS1 and NYS4 (kindly provided by Dr. Y. Charoenvit, N.M.R.I., U.S.A) than incubated with rabbit anti-mouse immunoglobulin and 10nm protein A-gold. Controls were carried out by incubation with normal mouse serum instead Mabs.

NYS1 recognize PyCS protein and produced intensive label in both blood schizont and merozoites. Schizontes showed gold particles all over its cytoplasm but very little or none at membrane level while merozoites contained strong label at apical side in structures that remember micronemes and rhoptrias. No significant membrane label was observed. NYS4 that recognize PySSP2 produced similar results on general distribution, however, the label observed on matures merozoites was not so strong at the apical portion as for CSP, but structures, possible micronemes were densely labeled. Ours findings confirm previous results where CS protein was demonstrate in invasive forms of blood stages malaria parasites and are the first report of PySSP2 in blood stage parasites.

Supported by CNPq and FIOCRUZ.

046

LOCALIZATION OF g-TUBULIN ON TACHYZOITES OF TOXOPLASMA GONDII

¹Melo, E. J. T. & ^1,2De Souza, W. 1. Lab. Biologia Celular e Tecidual, CBB,UENF, Campos-RJ, 2. Lab. Ultraestrutura Celular Hertha Meyer, Inst. Biofísica Carlos Chagas Filho, UFRJ,RJ

Toxoplasma gondii is an Apicomplex parasite that presents in the apical body position a complex structure known as conoid. The conoid is formed by microtubules disposed in rings. The conoid structure is involved response in the rotation and locomotion of parasite. Therefore, it plays an important role on the invasion process of the parasite into the host cells. However, little is known about the formation and dynamics of conoid microtubules during division of toxoplasma gondii. Our previous studies showed that a-tubulin is distributed in the anterior position and throughout parasite cytoplasm. Observation of slightly extracted and considered negatively stained tachyzoites it has been shown that a set of microtubules originate form the conoid, irradiating towards the posterior region of the protozoa. We decided to analyze the localization of g-tubulin, a tubulin only found in microtubule organizing centers, using immunofluorescence and immunocytochemistry. Tachyzoites of Toxoplasma gondii obtained from peritoneal cavities of infected Swiss mice were fixed with glutaraldehyde (0.25% for immunofluorescence or 2.5% for electron microscopy) and extracted with Triton X-100 (0.1-2%). Parasites were incubated with a monoclonal antibody against g-tubulin (1:50), and then with a fluorescein-labeled secondary antibody. Parasites were then observed in a Confocal Laser Scan Microscopy. For electron microscopy the parasites were placed on the poli-L-lisin-coated coverslip. The parasites were fixed and lysed by Triton X-100 (1%) in PHEM (60mM Pipes, 25mM Hepes, 10mM EGTA, 3mM MgCl₂) buffer. The tachyzoites were fixed for 20 minutes in each of the following solutions:2% glutaraldehyde, 0.1% tannic acid and 5% uranyl acetate. After fixation the specimens were dehydrated with acetone up to 100%. Critical point dying was performed using CO₂. Dried specimens were shadowed with gold and carbon using a sputtering evaporator device. Glass coverslips were removed and replicas were rinsed with water, mounted and examined in Zeiss EM900. Parasites were intensely labeled with anti g-tubulin antibodies in the specific anterior position of tachyzoites. The examination of replicas of parasites extracted under conditions of cytoskeleton stabilization and labeled with anti g-tubulin antibodies showed labeling of the conoid region. Taken together these observations suggest that the conoid of tavhyzoites of T. gondii can be a microtubule organizator center.

Supported by FINEP, CNPq, PRONEX, FENORTE

047

LOCALIZATION OF KINESIN IN GIARDIA LAMBLIA BY VIDEO-ENHANCED IMMUNOFLUORESCENCE MICROSCOPY AND CRIOULTRAMICROTOMY

Campanati, L.*, Monteiro-Leal, L.H.** & De Souza, W.*^,***

* Universidade Estadual do Norte Fluminense, Centro de Biociências e Biotecnologia, Laboratório de Biologia Celular e Tecidual. Av Alberto Lamego, 2000 Campos - RJ

** Universidade do Estado do Rio de Janeiro, Departamento de Histologia e Embriologia. Av Prof. Manoel de Abreu,48 3^o andar Maracanã, Rio de Janeiro - RJ

*** Universidade Federal do Rio de Janeiro, Laboratório de Ultraestrutura Celular Herta Meyer. Riode Janeiro - RJ

Kinesin is an ATPase that is involved in cytoplasmic movements in virtually all eukariotic cell. Kinesin movements have polarity, since it's a motor protein responsible for anterograde transport ("+" end - directed) along microtubules. This is a very important molecule in intracellular movements of vesicles. Attached to receptors on the vesicle membrane, this molecule transports the vesicles from the (-) to the (+) end of microtubules. In this work we used the video-enhanced immunofluorescence microscopy and transmission electron microscopy of immunolabeled cryosections to localize this molecule in the primitive eukariote Giardia lamblia. These techniques are ideal to localize molecules that occur in low amount in cells. Enhancing the acquisition time of the video camera, one is possible to obtain good images of low levels of light. In this case, the localization of kinesin by routine immunofluorescence microscopy was not possible, but with the help of video-microscopy we could observe that this molecule is found around the basal bodies of the flagella and along the ventral flagella of Giardia. Electron microscopy of cryosections first incubated with the antibody against kinesin and with gold-labeled antibodies confirmed the observations made by video-microscopy.

Supported by: FENORTE, PRONEX, FINEP, CNPq.

048

CHARACTERIZATION OF TUBULIN DIVERSITY IN GIARDIA LAMBLIA

Campanati, L*. & De Souza, W.*^,**

* Universidade Estadual do Norte Fluminense, Centro de Biociências e Biotecnologia, Laboratório de Biologia Celular e Tecidual. Av Alberto Lamego, 2000 Campos - RJ

** Universidade Federal do Rio de Janeiro, Laboratório de Ultraestrutura Celular Hertha Meyer.

Giardia lamblia is a world wide spread protozoan that causes enteric diseases in children and adults. The disease is characterized by intestinal discomfort and deficient absorption of nutrients. This cell is pearl shaped, contains four pairs of flagella and a distinct organelle that is responsible for its attachment to the epithelium of the host, the adhesive disc. These organelles, and the median body, are in part composed by tubulin. This molecule is important in the maintenance of cell shape and in cytoplasmic transport of organelles and metabolites and is found in many forms in eukariotic cells as a result of post-translational modifications or the expression of different genes in the host. In this work we used the confocal laser scanning microscopy and transmission electron microscopy of immunolabeled cryosections to characterize the tubulin diversity in Giardia. In this study we used antibodies that recognize alpha (a) tubulin, gamma (g) tubulin, tyrosinated tubulin and polyglicylated tubulin. The antibody anti-alpha tubulin labeled the flagella, median body and the adhesive disc and the antibody anti-gamma tubulin did not label the flagella neither the median body, but showed affinity with the adhesive disc. Tyrosinated tubulin was found in the flagella and in the median body. Two antibodies that recognize different degrees of tubulin polyglicylation were used. One of them recognizes the flagella and median body and the other recognized only the median body. These antibodies did not label the flagella of protozoa of the Trypanosomatidae and Trichomonadidae families.

Supported by: FENORTE, CNPq, PRONEX, FINEP.

049

STRUCTURE AND ULTRASTRUCTURE IN SCANNING ELECTRON MICROSCOPY OBSERVATION OF STYLONYCHIA PUSTULATA (MUELLER, 1786) EHRENBERG, 1835 (HYPOTRICHIA) FOUND AT WATER-TANK OF BROMELIACEAE FROM SUGAR LOAF, URCA, RIO DE JANEIRO.

Carneiro, T.C. de S. & Silva Neto, I. D. da

Laboratório de Protistologia, Departamento de Zoologia, IB/CCS, UFRJ, Rio de Janeiro 21941-590, Brasil.

The Bromeliaceae accumulates rain water among the leaves, that forming a reservoir, where maintain many organisms with a evident trophic activity.

The Protists ciliates are importants predator of bacteria and others microorganisms, they are finding in large number. The samples of water were collected from Bromeliacea Alcantarea geniculata, found at Sugar Loaf- Urca, Rio de Janeiro at about 200 high, the ciliate that we isolated is Stylonychia pustulata.

Stylonychia pustulata was cultivated in mineral water (Petrópolis) with grains of wheat with bark for development of bacteria.

The ciliate has ovoid body with 85-140 mm of length and 42-68 mm of width.

The somatic ciliature present three posterior cirri and meridional kinetids in the dorsal side, whose border are lightly convergents

In the ventral side, the right marginal has 30-33 cirri and the left marginal has 20-22 cirri.

The frontal cirri are constituting of one buccal cirrus, four frontoventral and three postorals.

The buccal ciliature present the oral zone delimited by the adoral zone of membranelles with approximately 41 membranelles, that occupy the anterior half of cell body.

The paroral membranelle is composing of one row of kinetossoma.

In the posterior left oral region are located one contractile vacuole.

The ciliate present one micronucleus on the left behind of the two ovoid macronuclei.

Stylonychia pustulata found for us has the same features from the European and African specimens.

Supported by CNPq, FAPERJ and CPEG-UFRJ.

050

STRUCTURE AND ULTRASTRUCTURE ASPECT OF UNCOMMON CILIATE (CILIOPHORA: HYPOTRICHIDA) FROM SOIL OF TIJUCA FOREST, RIO DE JANEIRO.

Passos, M.I.da S. & Silva Neto, I.D. da

Laboratorio de Protistologia, Departamento de Zoologia, IB/CCS, UFRJ, Rio de Janeiro, 21941-590, RJ., Brasil.

The Tijuca forest is located in a urban area at Rio de Janeiro. It was reforested with a variety of plant species. Its soil is rich with organic matter, and holds a variety of organisms including ciliates protozoans. We collected several soil samples and took it to the laboratory to let it dry, than little samples were stored in Petri dishes and rehidrated with distilled water. These were examined one or two days after, under a stereoscopic microscopic to isolated the ciliates. They are maintaining in culture in laboratory. We cultivated the ciliates trying differents media, the most successful was with grains of wheat. The ciliates were fixed for optical microscopic, whose impregnated agent was silver proteinate and scanning electron microscopic fixed with the follow solution by 40 min.: glutaraldehyde 2%, osmium tetroxide 1%, added cacodilated buffer 0,2 M. An interesting ciliate was observed, and several specimens were examined. Their size have in average 114-136 mm of length and 51-67 mm of width, their bodies are ovoid and ventrally flattened. The oral apparatus is located on the left anterior occupying 1/3 of the body. Adoral zone of membranelles is constituted by approximately 45 membranelles, and paroral membrane is organized with one kinetossoma ciliphire row in ectoplasmatic fissure, on the right side of the buccal region. The somatic ciliature is compound by six rows of bristles whose are confluent anteriorly of the body, lightly convex in the dorsal view. Frontoventral cirri closely in number of ten. The right marginal row have twenty-eight cirri and the left marginal row have twenty-one, approximately, confluent posteriorly of the body. Cirri transverse are seven forming a structure in V. They present fourteen macronuclei and at least four micronuclei. These ciliates are closed by the family Oxytrichidae, however, the number of nuclei is different from the genera belong to it, whose present often two to four nuclei. Structure studies of its ciliature and nuclear complex will be present revealed by specific optical microscopic techniques and scanning electron microscopic.

Supported by CNPq, FAPERJ and CEPG-UFRJ.

ULTRASTRUCTURAL STUDIES ON ENDOCYTOSIS IN LOWER TRYPANOSOMATIDS

Rosane M.S. Meirelles and Maurilio J. Soares

Departamento de Ultra-estrutura e Biologia Celular, Intituto Oswaldo Cruz/ FIOCRUZ. Av. Brasil 4365, Manguinhos. 21045-900 Rio de Janeiro, RJ, Brazil.

Several studies with Trypanosoma brucei, T. congolense, T. cruzi and Leishmania have demonstrated that these parasites are able to ingest proteins such as LDL, albumin and transferrin by receptor-mediated endocytosis and/or fluid-phase pinocytosis. Ultrastructural studies using proteins coupled to colloidal gold particles allowed the identification of the cell compartments related to the ingestion of these macromolecules. The proteins enter the cell through the flagellar pocket and can be observed inside vesicles and in a complex network formed by several cisternae and tubules similar to endosomes and lysosomes. However, little is known about endocytosis in other trypanosomatids, either digenetics or monogenetics. Thus, in the present study we have analyzed by Transmission Electron Microscopy (TEM) the ingestion of proteins in several species of lower trypanosomatids.

Crithidia deanei, Crithidia fasciculata, Blastocrithidia culicis, Leptomonascolossoma and Herpetomonas muscarum muscarum were maintained in LIT medium for 1-2 days, washed for 30 minutes in PBS, and incubated at 28⁰ C for 1 hour with gold-labelled albumin or transferrin (8-10 nm). Thereafter, the cells were fixed in glutaraldehyde and processed for conventional TEM according to a quick processing protocol developed in our laboratory. Epimastigote forms of Trypanosoma cruzi were used as a control, since the endocytic process in this parasite is already known.

Our initial results showed the absence of colloidal gold particles bound to the plasma membrane or inside the cells (either in vacuoles or specialized organelles). In some parasites, the marker could be detected inside the flagellar pocket, but it was not bound to the pocket and flagellar membranes, what could indicate the absence of receptors for these proteins. Endocytosis of the labelled proteins could be observed in the epimastigote forms of T. cruzi used as a control. Label was found bound to the flagellar pocket membrane and inside reservosomes and vesicles in the cell cytoplasm. Further studies are needed to elucidate whether in the lower trypanosomatids endocytosis plays only a minor role in the obtention of nutrients or is an ephemeral process in their life cycle (mean doubling time about 6 - 8 hours). It is possible that most nutrients can be obtained by diffusion through the plasma membrane.

This work has been supported by Capes, CNPq, PADCT/CNPq and FIOCRUZ.

052

EFFECT OF THE SUPERNATANT AND CELLULAR FRACTIONS ISOLATED FROM STREPTOMYCES ALBONIGER ON TRYPANOSOMA CRUZI ULTRASTRUCTURE

Lopes, A, Meireles, MNL*, Coelho, RRR, Branquinha, MH & Vermelho, AB.

Dept. Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, UFRJ.

* Lab. de Ultraestrutura Celular, Dept. de Ultraestrutura e Biologia Celular, FIOCRUZ.

Trypanosoma cruzi epimastigote forms were treated with three factions isolated from Streptomyces alboniger, a soil actinomycete. Fraction 1 was obtained by concentrating the culture supernatant through dialysis against polyethyleneglycol. The hydrophilic ( fraction 2 ) and hydrophobic ( fraction 3 ) phases were extracted from S. alboniger with detergent Triton X-114. Briefly, the myceliun was extracted overnight at 4º C with 2% Triton X-114 in 10 mM Tris Saline Buffer, pH 7,4. The insoluble material was removed by centrifugation at 37,000 g for 20 min. at 4º C. The hydrophobic and hydrophilic phases were separated with a 6 % sucrose custion (1:1,5 v / v ) and proteins and glycoconjugates were precipitated with acetone.

Ultrastructural analysis showed that all fractions induced alterations in the parasite mitochondrion, which became swollen. However the hydrophobic fraction followed by the fraction 1 induced the most severe alterations. Secondary effects were detected in chromatin distribution and in the number of reservosomes.

Since T. cruzi mitochondrion is a selective target for many anti- T.cruzi drugs, further studies will must be done in order to characterize the components of the extracellular and hydrophobic fractions from S. alboniger.

Supported by : FINEP-BID, CNPq, FUJB, PAPES-FIOCRUZ, CEPG-UFRJ.

053

STUDIES OF SUBCELLULAR LOCALIZATION OF RAB7 IN TRYPANOSOMA CRUZI:

Araripe, J.R.*; Leal, S.T.*; Cunha e Silva, N.L.**; de Souza, W.** & Rondinelli, E*.

* Lab. Metabolismo Macromolecular Firmino Torres de Castro, ** Laboratório de Ultraestrutura Celular Hertha Meyer - Instituto de Biofísica Carlos Chagas Filho, UFRJ, 21949-900, Rio de Janeiro, RJ.

The small monomeric GTPases of the rab subfamily are key elements of the machinery that controls membrane traffic in eukaryotic cells. Approximately 30 different rab genes have been identified in a variety of mammalian species and localized to many different intracellular organelles on both the endocytic and exocytic pathways. This suggest that each step of membrane traffic might involve a different rab protein acting as a catalyst, accelerating the fusion reaction between donor and acceptor vesicles promoted after v/t-SNARE complex assembly. The Rab7 protein is associated with late endosomes in mammalian cells and several results suggest that this protein is involved in the anchoring step of these vesicles during endocytosis. The rab7 gene of Trypanosoma cruzi (tcrab7)has already been sequenced and characterized in our laboratory. We obtained transformed cell lines stably expressing Rab7 protein (pTAG) and a similar protein but without the last three C-terminal residues (DCXC). This Rab7 mutant fails to associate with membranes. We used a polyclonal antibody raised against a synthetic peptide comprising amino acid residues localized in the C-terminal region of Rab7 in order to localize the protein by immunofluorescence microscopy. This antibody showed that Rab7 is located at the anterior region of the cell, near the kinetoplast and the bottom of the flagelar pocket; this structure is duplicated in dividing cells. The intensity of staining was the same for all cells lines studied, including wild type CL Brener.These results are in agreement with those described for Trypanosoma brucei (Field,H. & Field, M.C., 1997, J. Biol. Chem., 272:10498). We are currently studying the distribution of Rab7 by immunoelectronmicroscopy. We also obtained a single transformant clone that is partially knocked out for the rab7 gene (Araripe et al, acompanying abstract). Its morphological characterization showed alterations at the anterior region of the parasite when observed at electron microscopy. Further characterization of this transformant is in progress.

Supported by CNPq, FAPERJ, CAPES and FINEP

054

INTRACELLULAR TRAFIC OF CRUZIPAIN IN EPIMASTIGOTE FORMS OF TRYPANOSOMA CRUZI

Fonseca, P.C., Figueiredo, I.F. & Souto-Padrón,T.

Laboratório de Protozoologia I , Instituto de Biofísica Carlos Chagas Filho - UFRJ - CCS, Bloco G - Ilha do Fundão, Rio de Janeiro, RJ, 21949-900 - Brasil.

Cruzipain, the major proteinase of Trypanosoma cruzi, is present in all evolutive forms of the parasite. The enzymatic activity is developmentally regulated being 10 fold higher in epimastigotes than in other stages (Campetella et al., FEMS Microbiol. Lett. 67:145,1990). Biochemical evidences clearly suggested a lysosomal localization. Ultrastructural studies associated to immunocytochemical analysis showed the presence of cruzipain on the plasma and flagellar pocket membranes, free inside the pocket and in several structures involved in the endocytic process of the parasite (Soares et al, J.Cell Sci. 102: 157, 1992). According Cazzulo et al.(Mol.Biochem.Parasitol. 38: 41,1990) cruzipain does not contain mannose-6-phosphate tags suggesting an alternative sorting to the lysosomes. This study investigates the exposition of cruzipain on the cell surface of epimastigotes and its landing to cytoplasmic structures. Four-day-old culture forms of the T. cruzi Y strain cultivated at 28⁰C in LIT medium were used. Parasites were collected by centrifugation, washed in phosphate bufferd saline (PBS),pH 7.2, and then incubated for 30 min at 4⁰ C in Hank's medium without serum. The cells were then incubated in the presence of a polyclonal antibody against cruzipain, 1:200 dilution, and in the presence of a gold-labeled goat-anti rabbit antibody,1:100 dilution. Samples were harversted immediately after the incubation with both antibodies (T0) and after 5 (T1), 10 (T2) and 30 (T3) minutes of incubation at 28⁰C. All samples were fixed and processed to conventional transmission electron microscopy and to immunocytochemical procedures. Labeling of epimastigote forms incubated during 30 min at 4⁰ C showed the presence of gold particles associated to the cell body membane (few particles) and a lot of particles in the cytostome where they are associated to the membrane or on small bublles present in this region. A lot of labeled membrane profiles was observed suggesting a shedding process of cruzipain in the presence of the antibody even at 4⁰C. Five or 10 min after incubation at 28⁰ C gold particles were seen inside small vesicles and tubular structures in the region of the Golgi complex. Thirty minutes after incubation at 28⁰ C, gold particles were seen inside larger structures in the posterior regions of the parasite ressembling reservosomes. The analysis of the influence of drugs in the process of intracellular traffic of cruzipain are in progress.

Supported by PRONEX, FINEP, CNPq & CEPG-UFRJ

055

CHARACTERIZATION OF NEW GIANT PROTEINS ASSOCIATED WITH THE TRYPANOSOMATID CYTOSKELETON

Ricardo N. Gobbi; Munira M. A. Baqui; Júlio Pudles

Departamento de Parasitologia - Instituto de Ciências Biomédicas, Universidade de São Paulo. Caixa Postal 66208, São Paulo, 05508-900,

SP, Brasil.

In previous reports, we have identified and characterized megadalton proteins associated with the cytoskeleton of Phytomonas serpens, Leptomonas samueli and Crithidia l. thermophila. In this communication we have expanded these investigations to three new genera of trypanosomatids (Blastocrithidia culicis, Herpetomonas samuelpessoai and Leishmania tarentolae).

We have observed the presence of giant proteins associated with the cytoskeleton, by SDS-PAGE in these three genera. These proteins present different migration patterns with Mr. ranging from 900 to 2500 k. These results were confirmed by submitting the proteins to harsher denaturing conditions (using 2% SDS and 4M urea or 6% SDS).

Immunoblott and immunoprecipitation of these proteins metabolically labeled with [³⁵S] methionine, suggested that polyclonal antibodies raised against each of the proteins are genera specific.

These results indicate that giant proteins are ubiquitous cytoskeleton components of the Trypanosomatidae family and may play an important structural function at the flagellar pocket region.

Support by: FAPESP and CAPES

056

LOCALIZATION OF INTERLEUKIN-1b-CONVERTING ENZYME (ICE) EPITOPES IN LEISHMANIA (LEISHMANIA) AMAZONENSIS PROMASTIGOTES BY LASER SCANNING CONFOCAL MICROSCOPY

Moreira, M.E.C.^a , Balanco, J.M.F.^b , Pitaluga, A.N.^a, Mota, E.M.^c, Lenzi, H.^c, Barcinski, M.A.^b & Traub-Cseko, Y.M. ^a

^a Departamento de Bioquímica e Biologia Molecular, IOC, FIOCRUZ, Rio de Janeiro, RJ.

^b Departamento de Parasitologia, ICB, USP, São Paulo, SP.

^c Departamento de Patologia, IOC, FIOCRUZ, Rio de Janeiro, RJ.

We have previously shown that Leishmania (Leishmania) amazonensis promastigotes die by apoptosis when submitted to an in vitro heat shock in the presence of calcium ions (1). Susceptibility and protection to this type of cell death are gene-regulated conditions and are respectively related to the expression of ICE/ced-3 and bcl-2 gene families in diverse cell types (2). The ced-3 gene plays a major role in apoptosis induction in the nematode Caenorhabditis elegans and codifies an homologue product to the mammalian cysteine protease ICE (3). Considering these findings, we looked for ICE and bcl-2 products homologue proteins in L.amazonensis promastigotes in order to elucidate apoptosis pathways in this protozoan parasite. Heat-shocked and non-heat-shocked promastigotes were first incubated with a rabbit polyclonal antibody, directed against amino acids 390-404 of the human ICEa protein, and then with a secondary antibody against rabbit IgG conjugated to rhodamine. As an alternate strategy, parasites were incubated with a biotinylated ICE inhibitor (Biotin-Tyr-Val-Ala-Asp-acyloxymethylketone) and then with an avidin-fluorescein conjugate. Preparations were examined by Laser Scanning Confocal Microscopy. All parasites incubated with the ICE inhibitor displayed ICE epitopes distributed mainly in their cytoplasmic ground substance, differently from promastigote population incubated with the ICE antibody. In the latter case only a small porcentage of the parasites came up as positives. These results strongly suggest that these epitopes may be differently exposed in a heterogeneous population of promastigotes. We conclude that apoptosis induction pathways display some similarity between protozoa and mammalia. The localization of bcl-2 product epitopes as well as the distribution of ICE epitopes in heat-shocked parasites are current under investigation.

(1) Moreira et al., J. Cell.Physiol. 167: 305-313, 1996.

(2) Williams & Smith, Cell 74: 777-779, 1993.

(3) Thornberry et al., Nature 356: 768-774, 1992.

057

HEMOCYTES FROM UNINFECTED T. INFESTANS CAN PROTECT PARTIALLY MICE FROM TRYPANOSOMA CRUZI INFECTION

Levy, AM¹, Toledo Jr, RAS¹, Hoshino Shimizu,S² Lourenço AM¹, Pereira-Chioccola,VL¹ & Maifrino,Lbm¹

1. Inst. Dante Pazzanese de Cardiologia CP512; 2 Inst. Adolfo Lutz São Paulo

Hemocytes (HC) of uninfected T. infestans were previously shown to be recognized specifically by sera from patients with chronic Chagas disease in an indirect immunofluorescence assay (IFA), indicating that infective parasites and HC share common epitopes. Now we found that HC can partially protect mice from a challenge with T. cruzi. HC were collected from uninfected 5^th instar T. infestans nymphs with an anticoagulant solution and centrifuged. The cell sediment and also the supernatant mixed with alumen's adjuvant were inoculated i.p. in three groups of female A/Sn mice, three times at 10-15 days intervals. The first group of mice (HC) receveid 2, 3 and 4 x 10⁶ HC/ml, the second group (HL) receveid 188-200 mg% proteins of the hemolymph supernatant, and the third group which was a control group (C). was inoculated only with alumen's adjuvant (0.5mg/ml). In order to check the immune response, blood was collected from tail vein and their serum assayed by IFA using HC as antigen. Mice from the HC group exhibited high IgG antibodies to plasmatocytes and granulocytes. All animals were challenged intraperitoneally with 5 x10² bloodstream trypomastigotes (Y strain) 15 days after the last immunization. Parasitemia was monitored by Brenner method from 6^th just 15^th day after infection, time when circulating parasites were no longer observed in the blood. Parasitemia peaks of the HL and HC groups were observed one or two days after the C group. In these same period of time both HL and HC groups had significantly low parasitemia, i.e. about 63% lower in comparison to the C group. However the mortality shown by HC and HL groups differed 45 days after infection, being 14% and 43%, respectively, though being lower than 56% of the C group. Thus epitopes of hemocytes, which confer partial immunoprotection to the T. cruzi infection in mice require to be further identified and characterized.

supported partially by CNPQ

058

RABBIT CORNEA CELLS AS A MODEL FOR ACANTHAMOEBA PATHOGENICITY

Lopes, L.M.¹; Scaletsky, I.C.A².; Pedroso, M.Z.³; Foronda, A.S¹. & Affonso, M.H.T¹.

¹Departamento. de Parasitologia, ICB/USP; Av, Prof. Lineu Prestes, 1374 CEP 05508-900, São Paulo (SP): ²Disciplina de Microbiologia, EPM/UNIFESP; ³Disciplina de Gastroenterologia, EPM/UNIFESP

The free-living protozoa Acanthamoeba are implicated in severe pathologies for animals and humans. Several Acanthamoeba species have been described to cause severe keratitis associated with the use of contact lenses, and granulomatous amebic encephalitis (GAE) that occurs in immunodepressed individuals. Despite the increasing number of cases in the world, the pathogenic mechanisms of Acanthamoeba are not yet understood. This fact is due mainly by the lack of appropriate in vitro or in vivo models that could be used to study the pathogenesis of these organisms and even to improve either the early diagnosis or an efficient treatment of the Acanthamoeba keratitis.

Many attempts to achieve an animal model for cornea pathology have been unsuccessful. We are trying to use several cell lineages in order to establish a model to evaluate the pathogenic potential of Acanthamoeba isolates from environment and cornea. VERO, Hep2 and LLC-MK2 cells were used. After preliminary tests, we decided to use a cell lineage derived from rabbit cornea (SIRC cells).

SIRC cells were routinely maintained in Eagle plus Dulbecco medium and confluent cultures were harvested with trypsin. Cell suspensions were grown into 96 well microtiter plates (1x10⁵ cells/well) and incubated for 48 h at 37^oC with 5% CO₂. Confluent cells were then co-incubated with Acanthamoeba trophozoites (2.5x10⁵ cell/well) suspended in the same media, and the plates were incubated at 37^oC with 5% CO₂. Control wells contained SIRC cells without amoebas. After 24 hours, wells were rinsed with cold water, fixed in 2% formalin and stained with crystal violet. Cytopathic effect was evaluated by destruction of the cells monolayer. Preliminary results have shown that both cornea and environment isolates produced cytopathic effect on SIRC cells. Nevertheless, this effect from the cornea isolates was much more conspicuous than the environment ones. No cytopathic effects were observed with VERO, Hep2 or LLC-MK2 cells. Other environment and cornea isolates are now being tested. These results suggest that SIRC cells can be used as a suitable model for Acanthamoeba pathogenicity.

Supported by FAPESP and CNPq

059

EXPERIMENTAL TOXOPLASMOSIS AND IMMUNOSSUPRESSION. LEVELS OF PARASITEMIA AND HISTOLOGICAL ASPECTS

Guimarães, AC, Kawarabayashi, M, Nunes, EV, Ferraz, SN, Chiosini, CB, Okumura, M. & Tolezano, JE

Seção de Parasitoses Sistêmicas do Instituto Adolfo Lutz - São Paulo-SP

In this study, part of project on experimental toxoplasmosis and immunodeficiency, we evaluated the levels of parasitemia and histological injury in mice with and without immunossupression by strain of no forming cysts-Toxoplasma gondii. We constituted three types of experiments, all of them based on concentration of tachizoites of no cystogenic RH strain of T. gondii, at 1x10³, 3x10³ and 2x10⁵ parasites respectivelly. For all experiments, mice were divided into groups with immunossupression (ciclosporine and hydrocortisone), without immunossupression group and control groups. The observations and quantifications of parasites were made every 24 hours by examinations of blood collected from heart puncture and of peritoneal exsudate. The count of T. gondii was made in a Neubauer chamber. Each day, two mice of every group were sacified in order to do histological observations from heart, liver, spleen, lung and brain specimens. In tissues sections of these organs it was done an immunofluorescence reaction by using an anti-T.gondii. Independently of the number of inoculated parasite, we observed 100% of death, at time between 72 and 144 hours, among mice from experimental groups of all three types of experiments. The patent parasitemia was more precocious at about 24 hours in immunossupressed group. In peritoneum we observed a counting of T. gondii 10 times more elevated than in the blood from heart. The "systemic dissemination" and mortality rates were both more precocious, at about 24 hours, in immunossupressed groups. The control groups survived until the end of the experiments. The tissue section stained by HE showed a greater parasite aggression and extense necrotic areas in the nervous tissues of the immunossupressed groups. It was also showed the presence of T. gondii in capillary vessels with apparent obstruction. The presence of parasite in the histopatological lesions were confirmed by immunofluorescence reaction. The high virulence of RH strain of T. gondii, with great necrosis in the brain tissues and the fulminant evolution in acute phase were aggravated by immunossupressors activities.

060

TOXOPLASMA GONDII SURVIVAL AND MULTIPLICATION INTO CHICKEN THROMBOCYTES COCULTURED WITH LYMPHOCYTES.

DaMatta, R.A.^1&2 , Manhães DS, L.¹, Seabra, S.H.¹, & De Souza, W^1&2

¹Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, UENF and ²Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brasil.

To test the phagocytic ability of chicken thrombocytes (mammalian platelet analogs) we used Toxoplasma gondii and demonstrated that this parasite can actively invade chicken thrombocytes with no involvement of host cell phagocytosis (DaMatta et al., Mem. Inst. Oswaldo Cruz, Rio de Janeiro, 91, Suppl., 80). In order to determine whether or not Toxoplasma gondii can survive and multiply into chicken thrombocytes, interaction with thrombocytes in a 10/1 parasite/thrombocyte ratio was carried out for 1 hour, washed and further incubated to detect survival and multiplication of the parasite. Thrombocytes were separated from chicken blood by a Percoll cushion and further purified by adherence on coverslips; lymphocytes were recovered from the washes and kept in ice. Tachyzoites of T. gondii, RH strain, were obtained from mice infected 2 days before; peritoneal wash was centrifuged and the supernatant used. After the interaction, cells were washed (to remove parasites that did not invade cells) and cultured for 24, 48 and 72 hours in DMEM supplemented with 10% of fetal bovine serum in the presence of lymphocytes. Thrombocytes are very sensitive and to extend their culture period need soluble factors secreted by lymphocytes. After the interaction or the cultured time, cells were fixed with bouin, stained with Giemsa, dehydrated in acetone-xilol, mounted in Entellan and observed under a light microscope. Toxoplasma gondii could invade thrombocytes, as shown before; multiplication of the parasite could be detected, but the majority of the infected thrombocytes were found only with one parasite even after 72 hours. Macrophages present in the culture could also be infected, but the multiplication of the parasite was also low. Thrombocytes could serve as a Toxoplasma gondii reservoir. The non-multiplication of the parasite could be due to lymphocyte secretion products which, somehow, were tuning up the thrombocyte microbial system.

Supported by FINEP, FENORTE, PRONEX and CNPq.

CASES REPORTED OF THE INTESTINAL CRYPTOSPORIDIOSIS COMPLICATED BY DISSEMINATED CYTOMEGALOVIRUS INFECTION.

Pinto, M.; Rivera, L.; Ballester, D.; & Lauro, L.

Hospital General de Agudos Parmenio Piñero. (GCBA). Av. Varela 1307 (1406), Buenos Aires, Argentina. Phone: 541- 631-8100 ext.1400. Fax: 541- 806-5900.

Cryptosporidium sp. is an enteric apicomplexan capable of causing moderate to severe diarrehal illness in immunocompetent individuals and life-threatening disease in immunocompromised subjects.

It has been called to attention by it&acute;s significantly high incidence in human patients with acquired immunodeficiency syndrome ( AIDS ).

The purpose of this report is to describe the clinical features and anatomic findings in patients with intestinal cryptosporidiosis complicated by disseminated infection by Cytomegalovirus ( CMV ), with extensive involvement of the bowel and colon and adherence of bacteria to damaged colonic epitelial cells.

Ten patients had been studied during 1996.The organisms were identified by light microscopy in a biopsy specimen from the duodenum and stool samples. The exact source from wich humans acquired cyptosporidial infection is unknown.

Transmission from animals is a possibility, but remains unproved if oral ingestion of an unidentified infective stage of the organism may be involved.

In eight of the ten reported cases of cryptosporidiosis, preexisting immunocompromise appeared important in the genesis of infection.The clinically important immunosuppression developed during the course of the disease is suggested by the prolonged course and disseminated nature of infection by CMV.

Of special interest is the initial detection of a low titer of antibody for CMV followed by a significant increase after the development of pneumonia due to this agent. This suggests that the virus was latent early in the clinical course of the cryptosporidiosis and did not play a role in the pathogenesis of the intestinal disorder.

It is interesting to speculate that pulmonary or other macrophages laden with CMV may have migrated to the bowel from other sites.

Infection with CMV has also been associated with persistent diarrehea. The epitelial cells and/or the mucosal and submucosal enteric capillaries and venules are involved.

Ulceration of the mucosa develops possibily as a result of local ischemia produced by focal vascular lessions.

062

EXPERIMENTAL INFECTION OF TOMATOES (LYCOPERSICON ESCULENTUM) BY TRYPANOSOMATIDS OF SEVERAL GENERA ISOLATED FROM PHYTOPHAGOUS INSECTS.

Cavazzana Jr, M.; Tano, M. S.; Catarino, L.M.G.M.; Baccan, G.C.; Jankevicius, J.V. & Itow Jankevicius, S. Universidade Estadual de Londrina, Centro de Ciências Biológicas Departamento de Patologia Geral. Caixa Postal 6001, CEP 860510-900, Londrina-PR.

The natural infection of vegetable species by trypanosomatids has been described since a long time ago in a diversity of plant species and phytophagous insects such as Nezara viridula, , Euchistus herus, Piezodorus guildinii (Pentatomidae) Leptoglossus zonatus (Coreidae). These insects are could be considered natural vectors of these protozoa in the nature.

In this study we studied the possibility of trypanosomatids isolated from phytophagous insects to develop in the tomato fruits. These strains were previously characterized by enzymatic profile of Ornithine-Arginine cycle and showed a profile similar to that of the genera Herpetomonas, Crithidia, Leptomonas, and Phytomonas. Ten microliters (10⁶ cells previously washed with 0.15M NaCl solution) were inoculated in tomatoes with the aid of needle and syringe. The needle orifice was covered with paraffin and the fruits incubated at 28⁰ C for 10 days . After that inoculate fruits were observed daily and the trypanosomatids quantified in Neubauer chamber.

All the strains were able to grow in the tomatoe fruit suggesting that those insects are vectors.

Financial support: CNPq, CAPES and CPG-UEL.

063

EFFECTS OF SERIC IRON LEVELS ON THE EVOLUTION OF THE EXPERIMENTAL INFECTION BY TRYPANOSOMA CRUZI

Amaral, J.F.¹, Oliveira, A.P.C.¹, Cortes, D.F.¹, Silva, M.E. ², Bahia, M.T.¹, Afonso, L.C.C.¹ & Pedrosa, M.L.¹

¹Depto. Ciências Biológicas, NUPEB, ICEB, ² Depto. Alimentos, Escola de Nutrição, Universidade Federal de Ouro Preto, Morro do Cruzeiro, 35400-000, Ouro Preto

The nutritional status of the host can influence the host-parasite relationship. Iron ions play an important role in several pathogenies. In this study we evaluated the effects of iron deprivation or supplementation on several aspects of the infection of swiss mice by the Y strain of T. cruzi. Thirty days old male mice were kept in stainless steel cages and fed a semi-purified diet with or without ferrous sulfate. Animals were divided into four groups: the control group (C), which received iron containing diet, a group which received control diet plus intraperitoneal injections of iron dextran (ID), a third group fed on an iron-deficient diet (NI), and a group which received the iron deficient diet plus intraperitoneal injections of desferrioxamine (DF). Fifiteen days after treatment onset part of the animals were inoculated with 500 blood stage forms of T. cruzi. Animals were sacrificed two weeks later. Iron serum levels of infected animals were decreased in groups NI and DF and increased in group ID when compared to the control group. Hemoglobin levels were also decreased in infected animals from groups IN and DF. Infected animals from the DF group showed a decreased spleen size when compared to the other groups. Parasitemia and associated mortality were higher in animals from the ID group while animals from the DF group showed a marked decrease in these parameters. Regarding the levels of IL-4 and IFN-g in Concanavalin A stimulated spleen cell cultures no differences were detected amongst the various groups whether the animals were infected or not. Thus, it appears that the alterations observed in the parasitemia and mortality may not be related to alterations in the host immune system but rather be associated with the availability of iron for the parasite.

Financial Support: FAPEMIG, CNPq/UFOP

064

THE FATE OF EPIMASTIGOTE FORMS OF TRYPANOSOMA CRUZI AFTER INTRAVENOUS INJECTION IN MICE. AN ULTRASTRUCTURAL STUDY.

Umekita, L.F.; Carneiro, S.M. & Mota, I.

Laboratório de Imunopatologia e Laboratório de Biologia Celular, Instituto Butantan. Av. Vital Brasil, 1500, São Paulo, 05503-900, SP, Brasil.

The non-infectivity of epimastigote forms of T.cruzi is supposed to be due to the ability of normal serum of many species to lyse these parasite forms. However, lysis is not the only mechanism to prevent infection by epimastigote forms since they are also non infectant in mice whose serum does not lyse them. Recently, we reported that when injected in mice epis are rapidly cleared from circulation by a non-lytic mechanism involving platelets and the alternative pathway of complement activation.

We report here the fate of 2x10⁸ epimastigote forms injected in mice. The lung, liver and spleen were collected and studied by histological and ultrastructural techniques. Beside liver and spleen the lung was an important site of clearance. In this organ the parasites were seen in close interaction with platelets, obstructing capillaries, or in the cytoplasm of phagocytic cells, in different stages of destruction.

We suggest that epimastigotes are non-infectious in mice because they activate the alternative pathway of complement, adhere to platelets through C3 fragments receptors that facilitate their removal and destruction by phagocytic cells.

065

EXPERIMENTAL MODIFICATIONS OF THE NATURAL TRANSMISSION OF TRYPANOSOMA CRUZI BY THE VECTOR

Heide, B.S.; Schaub, G.A.

Department of Special Zoology and Parasitology, Ruhr University, D-44801 Bochum, Germany

Investigating the modifications of the natural way of transmission of Trypanosoma cruzi to mammals, intradermal or subcutaneous injection of isolated T. cruzi and faeces of the vector into C57/Bl6 mice caused an initial retardation of the development of the parasite in the vertebrate host in comparison to injections without faeces. Simulating natural transmission led to very different results: Triatoma infestans with or without salivary glands were allowed to feed on the backs of C57/Bl6 mice. T. cruzi "Chile 5" were applied surfically to the area of the bite, using parasites in faeces of the bug, infested or free of bacterial symbionts, or PBS only. Whereas transmission of Leishmania is supported by the sandfly saliva (1), T. cruzi development does not seem to be affected by saliva of triatomines or different components of the faeces. In other experiments we injected different doses of T. cruzi (10 to 10⁴) into nude mice establishing a scale of disease development. Comparing the "natural transmission experiments" with these results we found that 50-500 T. cruzi invade the vertebrate host through the puncture of the mouthparts. The high rate of morbidity in our experiments (70 to 100%) shows that this way of infection is - contrary to other results (2) - not at all unlikely. Using additional T. cruzi strains we can find whether or not these differences are caused by the parasite.

(1) Theodos CM, Ribero JMC, Titus RJ (1991) Infect Imm 59, 1592-1598.

(2) Soares VA, Marsden PD (1986) Rev Soc Bras Med Trop 19: 165-166.

066

EFFECTS OF THE INFECTION BY TRYPANOSOMA RANGELI AND BEAUVERIA BASSIANA ON RHODNIUS PALLESCENS.

Agudelo ULA, *Moreno MJ.

*Department of Biology, University of Antioquia, A.A. 1226, Medellín, Colombia.

Chagas disease caused by Trypanosoma cruzi and transmitted by Hemipterous from Reduviidae family affect nearly 18 million people in Latinameric. In Colombia nearly 1.6 million people is affected. Trypanosoma rangeli shares vectors and hosts with T. cruzi, and acts as natural control of Triatomines, being innocuous to the vertebrates. Among the main vectors of T. rangeli, R. prolixus and R. pallescens stand out. This last one has a great epidemiologic importance, for its great capacity of domiciliation. This species has been found infected with T. cruzi and T. rangeli in three different department of Colombia: Bolivar, Sucre and Antioquia. Also, it is known that some fungi are pathogenic to the triatomines, standing out Beauveria bassiana.

In the present work, the strain SO-28 of T. rangeli isolated from R. pallescens, and the strain INRA-297 of B. bassiana, isolated from Hemipterous were used to carry out infections in R. pallescens. Mixed infections were carried out infecting insects with T. rangeli, once parasitism with the fungus was proved.

The percentage of infection of R. pallescens with T. rangeli was 100%. Morphologic alterations were detected in salivary gland level as regards to size, damage in tabique and less coloration of salivary gland. Malformation of thorax and proboscis were observed and difficulty to relase cuticle during molt was significantly different between infected (6.25%) and control (0%) insects.

During the follow up of parasitism, a 38.75% mortality was observed in R. pallescens infected and only 5% was found in controls. This infection influenced also the percentage of molting, beings significantly greater in controls (98.30%) than in infected insects (83.97%). Infection by T. rangeli modified the alimentary habits of the R. pallescens, making infected insects to take more time in search of food, with values between 9.2 to 24.4 days, contrasting with 0 to 14.9 days for controls.

As regards to the infection with B. bassiana, a mortality of 95% was observed between the days 2 and 20 postinfection, with a percentage of sporulation of 68.42%. In mixed infection the same percentage of mortality was observed.

R. pallescens showed to be very susceptible to the infections by T. rangeli and B. bassiana being the fungi more pathogenic than parasit. The simultaneous presence of T. rangeli and B. bassiana does not seem to have a synergic effect on the R. pallescens.

067

ESTIMATED COST OF A TRYPANOSOMA VIVAX OUTBREAK ON BROOD CATTLE IN THE PANTANAL (BRAZIL)

Seidl, A.F.^1,2, Silva, R.A.M.S.¹, Abreu, U.G.P. de¹, and Pellegrin, A.O.¹

¹Researcher, Centro de Pesquisa Agropecuária do Pantanal (CPAP/EMBRAPA), Rua 21 de Setembro, 1.880, CX Postal 109, CEP 79320-900, Corumbá, MS, Brasil.² Consultant, PROMOAGRO — IICA/BID.

Abstract: In an outbreak on 7 ranches in the northern subregion of Poconé, Typanosoma vivax was identified in the Brazilian Pantanal for the first time in 1995. The impact of T. vivax on African cattle ranching has been profound and its arrival in the Pantanal is cause for great concern. T. vivax infection in cattle results in abortions, low productivity and death. We estimate the financial impact of the first outbreak of Trypanosoma vivax in the Pantanal in order to provide a notion of the potential influence of the disease and an analytical basis for future analyses. Since cattle and calves represent investments to the rancher and the impact of a T. vivax is not necessarily concurrent with either investment or infection, a present valuation of the costs of the disease was appropriate. The total estimated present value of the 2,222 brood cows is US$855,470. About 34.5% of the brood cows were infected with the disease. The estimated cost of the outbreak is the sum of the present values of mortality, abortion, and productivity losses and treatment costs, or US$32,631 (US$14.68/brood cow, US$4,662/ranch, 4% of total value). The cost of fertility losses generated the greatest proportion of the estimated costs of the disease in our calculations (2/3 of total). Treatment costs created the second greatest source of costs to the ranchers (1/4 of total). Had the outbreak gone untreated, the estimated losses would have exceeded US$140,000 (US$63.76/brood cow, US$20,239/ranch, 17% of total value). Abortion and brood cow mortality prior to treatment costs were approximately 4% of the total, each. Diagnosis and treatment of the disease is cost effective. Outbreaks will likely increase in frequency and magnitude in the future warranting further research into potential control strategies for trypanosomes in the Pantanal and the Bolivian lowlands.

068

EFFECTS OF LITHIUM CHLORIDE ON CELL GROWTH OF HERPETOMONAS MUSCARUM MUSCARUM AND BLASTOCRITHIDIA CULICIS.

Carolina N. Spiegel & Maurilio J. Soares

Departamento de Ultra-estrutura e Biologia Celular. Instituto Oswaldo Cruz / FIOCRUZ. Av. Brasil 4365 Manguinhos. 21045-900 Rio de Janeiro, RJ. Brazil.

It is known that lithium ions induce alterations in biological systems, including apoptosis and interferences with cell proliferation and differentiation. It has been proposed that these ions hinder the DNA replication. A recent study showed that treatment with 50 to 200 mM of LiCl inhibited cell growth and differentiation of epimastigote forms of a Trypanosoma cruzi-like protozoan, with morphological alterations in the nucleus and kinetoplast-DNA.

In this study we analyze the effects of LiCl on cell growth and ultrastructure of Herpetomonas muscarum muscarum and Blastocrithidia culicis. The first undergoes a cell differentiation process similar to that which occurs in Trypanosoma cruzi. B. culicis may represent also a non-pathogenic model, since it lives under the epimastigote form. Furthermore, it presents endosymbiont bacteria in the cytoplasm, which accounts for a third kind of DNA to study.

H. m. muscarum and B. culicis were incubated for two days at 28ºC with 10, 50, 100 or 200 mM of LiCl added to the culture medium (LIT supplemented with 10% fetal calf serum). Aliquots were obtained at 24 hour intervals and cell growth was estimated by cell countings with a Neubauer chamber. After 48 hours the cells were processed for routine transmission electron microscopy (TEM).

Incubation of these protozoa with LiCl resulted in growth inhibition at all tested concentrations, in a dosis-dependent manner. Observation of the treated cells by TEM showed no signs of morphological degeneration. In H. m. muscarum the nuclei lost their peripheral heterochromatin and appeared filled with an homogeneous matrix. In B. culicis, incubation with LiCl induced the formation of several lipid droplets. When cells were incubated for 48 hours in the presence of the drug, then washed with PBS and allowed to grow in lithium-free medium, growth could be restored. Staining the cells with the vital dye Erythrosin B showed that most cells were viable. Our data suggest that LiCl treatment is arresting the cell division process in these parasites. Further studies will be carried out to analyze the effects of lithium ions on the differentiation process of Herpetomonas muscarum.

This work has been supported by CNPq, PADCT/CNPq and FIOCRUZ

069

CONTRIBUITION FOR THE ISOLATION, CULTIVATION AND MAINTENANCE OF THE FREE-LIVING AMEBAS.

Giazzi, J.F.; Buainain, A.; Rosa, J.A.da; Martini, A.S.; Belda Neto, F.M.; Martinez, I. & Fernandes, M.Z.T.

Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista - Caixa Postal 502, Araraquara, 14801-902, SP, Brasil.

INTRODUCTION: Free-living amebas are the most recently discovered protozoa there can produce harmful and lethal efects for the human life.They can invade the Central Nervous System and others organs, occasioning death or permanent incapacity (MARTINEZ, 1985).

MATERIAL AND METHODS: technics of direct examinations and culture are utilized, employing the Pavlova's Medium, Pavlova gel, solid Pavlova, Brewer and Diamond, in tubes and microculture between lamina and cover sleep ( in humidity chamber) samples of waters, dusts, sands and nasofaringean secretions of various origen were examined.

RESULTS: The obtained results were the meeting of free-living amebas in the analysed samples. In the one hundred and fifteen samples of investigated waters, amebas of the Acanthamoba genus were revealed in fourteen samples, and amebas of the Naegleria genus, in nineteen samples. In twenty three samples of analysed dusts, twenty positive occurrences of Acanthamoeba sp. amebas and nive occurrences of Negleria sp. amebas were found. We found for samples of both amebas of Acanthamoeba genus and amebas of Naegleria genus in the five samples of sands we analysed. Finaly, in eight samples of nasal secretions examined, amebas of Acanthamoeba genus were found in four of them.

CONCLUSION: The investigation is concluded alerting to the high diffusion of the reported protozoa in the nature and detaching the role represented by the Culture Mediuns used in the experiment, mainly by the performance of the Pavlova gel Culture Medium, that showed itself excellent to the isolation, development and maintenance of strains of the free-living amebas.

070

AXENIC CULTIVATION OF LEISHMANIA AMAZONENSIS AMASTIGOTE-LIKE STAGES

Moitinho^#, M L R, Pral, E M F, Teixeira, V R, Milder, R V & Alfieri, S C

Depto. de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, CEP 05508-900, São Paulo, SP, Brasil; ^#Depto. de Análises Clínicas, Centro de Ciências Biológicas e da Saúde, Universidade Estadual de Maringá, R. Colombo 3690, CEP 87020-900, Maringá, Paraná

Axenic amastigotes of Leishmania, i.e., those adapted for growth in cell-free medium, provide a source of parasites uncontaminated by the cellular, tissue-derived or fluid phase components present in animal lesions or persistently infected cell cultures. Amastigotes from several Leishmania species have been axenized, but they are often unavailable to other investigators. We have adapted Leishmania (L.) amazonensis LV79 (MPRO/BR/72/M1841) for axenic growth as amastigotes by progressively increasing the incubation temperature. Promastigotes in the fifth subculture at 22^oC in DL-15 medium were transferred to Schneider's Drosophila medium containing 20% (v/v) of heat-inactivated fetal bovine serum and were subcultivated 6 times at 31^oC, and then 10 times at 32^oC. At this stage, cultures still contained large numbers of flagellated stages, and were transferred to modified UM-54 medium, pH 6.2. Several subcultures were required in UM-54 medium at both 32 and 33^oC, untill growth of amastigote-like stages at 34^oC was successfully obtained. Growth curves of axenized amastigotes exhibited typical log and stationary phases, with doubling times varying from 11 to 13 hours. Growth rates were similar in absence of supplementary hemin and in medium prepared at pH 5.7. Axenic amastigotes could be serially maintained at pH 5.35, but their growth at pH 5.0 was difficult to sustain. At this stage, axenized parasites were similar to amastigotes in that they were ovoid and lacked a free flagellum. By transmission electron microscopy, axenic amastigotes displayed a very short flagellum, and were endowed with relatively few lysosomal-like, amastigote-specific organelles, the megasomes.

Proteinase activities, known to be developmentally regulated in L. mexicana, L. pifanoi and L. amazonensis, were also examined. In gelatin gels, both types of amastigotes displayed similar proteinases, but comparatively, total azocasein hydrolysing activity was 2.7 times higher in lesion-derived amastigotes. Axenic amastigotes were infective to BALB/c mice and avidly taken up by J774 cells. When placed at 34^oC in UM-54 medium, parasites recovered from animal lesions or cell cultures rapidly started to multiply, with doubling time values similar to those of parasites continuously kept in cell-free cultures.

Work supported by funds from FAPESP and CNPq (Brasil)

PARTIAL CHARACTERIZATION OF AXENICALLY CULTIVATED Leishmania braziliensis AMASTIGOTES

Balanco, J M F, Pral, E M F, Da Silva^#, S, Bijovsky, A T, Mortara^#, R A & Alfieri, S C

Depto. de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, CEP 05508-900, São Paulo, SP, Brasil; ^#Depto. de Microbiologia, Imunologia e Parasitologia e Centro de Microscopia Eletrônica, Universidade Federal de São Paulo, R. Botucatu 862, CEP 04062-040, São Paulo, SP, Brasil

Leishmania (Viannia) braziliensis (MHOM/BR/75/M2903) was adapted for growth at 34^oC and serially maintained as amastigotes in modified UM-54 medium, with growth curves exhibiting typical log and stationary phases. In late passages, parasite growth took place in absence of supplementary hemin and was unaffected when initial medium pH was adjusted between 5.4 and 6.3. Under phase contrast light microscopy and Nomarski differential interference microscopy, axenic amastigotes appeared round to ovoid, were immotile and lacked the extracellular flagellum. The absence of the paraflagellar rod (PFR) in the axenized amastigotes was confirmed by transmission electron microscopy, and lack of reactivity with monoclonal antibody 1B10 in both western blots and immunofluorescence microscopy. In western blots, the antibody recognized a 70/72 kDa doublet in Trypanosoma cruzi epimastigotes, and two, 70/74 kDa related proteins in L. braziliensis amastigotes. Immunoelectron microscopy of cytoskeletons of flagellated forms of T. cruzi and L. braziliensis indicated positive reaction of the antibody with the PFR. Axenic amastigotes and promastigotes were shown to differentially express surface components. Surface ¹²⁵I-labeling experiments identified promastigote-specific components (>100, 74, 45/47, and 28 kDa), and at least one, a 76 kDa polypeptide was specific for the amastigote stage. While axenic amastigotes were agglutinated by both peanut (PNA) and Lens culinaris (LCA) agglutinins (respectively at 50 and 12.5 mgml), promastigotes were not agglutinated by PNA and agglutinated with LCA at concentrations of 100 mg/ml and higher. Axenic amastigotes infected both J774 and rat bone marrow-derived macrophages. At multiplicities of 5 and 10 parasites per host cell, respectively 46.2 and 62.6% of marrow macrophages were infected at the end of 24 hours. By 48 hours, values dropped to 22.4 and 34.0%, respectively. Axenic amastigotes were avidly taken up by J774 cells, from which numerous organisms could be recovered 48 hours later. When placed at 34 ^oC in UM-54 medium, isolated amastigotes continued to multiply, thus indicating that viability was retained within the host cell.

Work supported by funds from FAPESP and CNPq (Brasil)

072

EFFECT OF PROTEOLYTIC INHIBITORS DURING HERPETOMONAS SAMUELPESSOAI DIFFERENTIATION.

Santos, A^.,.L.S.; Angluster, J. and Soares, R.M.A^,.

Departamento de Microbiologia Geral, Instituto de Microbiologia Professor Paulo de Góes (IMPPG), Universidade Federal do Rio de Janeiro (UFRJ), 21944-970, Cidade Universitária, Rio de Janeiro, Brasil.

The regulation of differentiation programs remains one of the central unresolved issues in modern biology. One significant limitation to the study of differentiation lies in the very nature of the process itself (Levenson & Housman, 1981). Trypanosomatids of the genus Herpetomonas are monoxenic parasites of insects which present promastigote, paramastigote and opisthomastigote in their life cycle, thus can be considered a excellent model for the studies of cell differentiation as well as morphological and physiological changes. The occurrence of a "leishmaniasis-like" infection caused by Herpetomonas in a patient infected with HIV (Dedet et al., 1995) poitend out the importance of the studies of these genus. In order to gain insight into the role of proteases in Herpetomonas samuelpessoai differentiation, cultures were grown in chemically defined medium (Roitman et al., 1972) with or without 4% of dimethylsulfoxide (DMSO) for 48 hours at 28⁰C in the presence of metallo (1,10-phenanthroline, 1 and 10mM) and cysteine (antipain, 1 and 10mg/mL) protease inhibitors. The growth was evaluated by counting the cells in a Neubauer chamber and the effect on cellular differentiation was determined by the percentages of cell morphological types in Giemsa stained smears. The results showed that the cell differentiation process, as well as the cell number are inhibited by antipain and 1,10-phenanthroline in a dose-dependent manner, suggesting that both cysteine and metalloproteinases might play a role in this process.

Financial Support: CNPq, CEPG-UFRJ, FINEP.

073

EFFECTS OF 1,4 DIAMINO-2-BUTANONE AND DL-a-DIFLUOROMETHYLORNITHINE IN ENTAMOEBA HISTOLYTICA AND ENTAMOEBA OSHKOVISKII.

Linhares ABR¹, Vannier-Santos MA², Yarlett N³ and Silva-Filho FC¹

¹ Laboratório Biologia da Superfície Celular and ² Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; ³ Haskins Laboratories, Pace University, NY

DL-a-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) antagonist and 1,4-diamino-2-butanone (DAB), a putrescine analogue were used to inhibit amebae polyamine biosynthesis. Polyamines play a pivotal role in cell growth and differentiation, regulating replication, transcription and protein synthesis. Entamoeba moshkovskii is a free-living protozoan used as an experimental model in parasitology research because of its similarity with the pathogenic Entamoeba histolytica. E. moshkovskii and E. histolytica were cultivated in the axenic TYI-S-33 medium supplemented or not with 20mM DAB and 30mM DFMO. Viable, adherent microorganisms were fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1M cacodylate buffer, pH 7.4 for 2h, and post-fixed with 1%OsO₄ in 0.8% potassium ferricyanide in the same buffer. Inibition of E. histolytica and E. moshkoviskii growth by DAB and DFMO were determined at 12h or 24h intervals, respectively. Ultrastructural observations of DAB-treated protozoa revealed the presence of a membranous system resembling endoplasmic reticulum cisternae, sometimes associated with glycogen granules and large amounts of ribonucleoprotein (RNP) helices which culminated with the formamtion of chromatoidal bars. However DFMO-treated protozoa presented electrondense granulous material within large vesicles. These data indicate that polyamines play important roles in the metabolism of both E. histolytica and E. moshkovskii.

Supported by FINEP, CNPq, PRONEX, FAPERJ and CAPES.

074

NUTRITION REQUIREMENTS OF LEPTOMONAS sp ISOLATED FROM PHYTOPHAGOUS INSECT.

Reuter-Filho, A.¹; Da Luz, O. J. D.¹; Seixas, A. S. S.¹; Miranda, A. M. M.¹; Cordeiro, N. S.² ; Manaia, A. C¹.

1.Dept° de Ciências da Saúde, CCBS - Universidade Federal de São Carlos (UFSCar), C.P. 676, São Carlos, 135665-905, S.P.; 2.Dept° de Parasitologia, IB-Universidade Estadual de Campinas (Unicamp), C.P. 6109, Campinas, 13083-970, S.P., Brasil.

We have previously reported the characterization of a trypanosomatid isolated from Leptocoris trivittatus, as Leptomonas sp (Manaia et al, Mem. Inst. Oswaldo Cruz, 90 Suppl. I: 260, 1995). The present study shows its nutritional requirements. Experiments were carried out in a defined medium described for H. samuelpessoai (Roitman et al, J. Protozool., 19: 346-349, 1972) and growth requirements were identified by omission of components of this medium. Experimental media were distributed 5.0ml per 18X180mm, screw-capped tubes. Each tube received 0,2ml of a 72-hr culture grown at 28C. Growth was measured by optical density using a spectrophotometer at 540nm and by counting the cells in a Neubauer chamber after 96-120-hr incubation at 28C.

The defined medium por H. samuelpessoai is also suitable for growth of Leptomonas sp from Leptocoris trivittatus. Glucose, fructose, maltose, sucrose were the best carbon sources at 28C. Sucrose was used thereafter. In the absence of sugars, reasonable growth was observed. Leptomonas sp required hemin, purine, the usual amino acids and vitamins requirements of insect trypanosomatids.

075

TRYPANOSOMA VIVAX IN SOUTH AMERICA: BIOMETRICAL DATA

Dávila, A.M.R.^{1, 2} ; Ramirez, L.^1,2 & Silva, R.A.M.S¹

¹Laboratório de Ecopatologia, EMBRAPA/Centro de Pesquisa Agropecuária do Pantanal (CPAP), Rua 21 de Setembro, 1880, 79320-900, Corumbá, MS, Brazil. ² UFMS/CEUC/DAM, Corumbá-MS; Brasil. Author for correspondence. alrida@cpap.embrapa.br.

Trypanosoma vivax has been reported in South America since 1919 (Leger & Vienne, 1919). A half century later, using an indirect fluorescent antibody test with a South American T. vivax as antigen, Wells et al. (1977) found positive sera of cattle from several South American countries (Colombia, Equador, Peru, Brazil-Mato Grosso and Paraguay). According to Hoare (1972), South American T. vivax has a range from 16mm to 26.5 mm in length. A more recent review showed that South American T. vivax varies from mean lengths of 15.86mm to 23mm in bovines from Bolivia (Santa Cruz department) and French Guyana, respectively (manuscript in preparation). Shorter forms were found in the Pantanal region of Brazil and Bolivian lowlands. Fairbairn (1953) showed that shorter forms would be related to the acute disease observed in West Africa. This observation has been considered by South American researchers, but further studies should be done to clarify the relationship among morphometrics and virulence of T. vivax.

Mean length of Trypanosoma vivax isolated from bovines in South America

Country (author) Mean length (mm) Country (author) Mean length (mm) French Guiana
(Leger & Vienne 1921) 22.5 Suriname
(Nieschulz & Frickers 1938)
(Nieschulz 1939) 21.0
21.5 Venezuela
(Tejera 1920)
(Fernandez 1931)
(Kubes 1944) -
-
22.0 Brazil
(Shaw & Lainson 1972)
(Silva et al. 1996)
(Silva et al. 1997) 22.77
18.73
18.10 Colombia
(Plata 1931) 21.0 Bolivia
(Silva et al. 1996)
(Silva et al. 1997) 15.86
17.37

076

ABSENCE OF IN VITRO MULTIPLICATION OF TRYPANOSOMA RANGELI WITHIN A MURINE PROMONOCYTIC CELL LINE

Eger, I.; Grisard, E.C. & Steindel, M.

Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Catarina. Caixa Postal 476, Florianópolis, Santa Catarina - Brasil. E-mail: ccb1mst@ccb.ufsc.br

Trypanosoma rangeli is a hemoflagelate parasite of man, domestic and sylvatic mammals and triatomine insects in Central and South America. In contrast to the pathogenic T. cruzi, it is harmless to the mammalian host but pathogenic to the vector. T. rangeli life cycle in the invertebrate host is well understood. On the other hand, there is little information about its cycle in the vertebrate host. In order to study T. rangeli in vitro infection, we used a promonocytic cell line and two distinct parasite strains. The mice promonocytic J774G.8 cell line was cultured in DMEM medium pH 7.4 supplemented with 10% FBS on circular glass coverslips. T. rangeli strains (Choachi and SC-58 B1 clone) were harvested from cultures in Grace's medium, washed in PBS and ressuspended in DMEM with 5% FBS. Cells were incubated with 10⁷ parasites (1:10 ratio) for 24 hours at 37^oC on 5% CO₂. Thereafter, slides were washed with PBS to remove free parasites and reincubated with DMEM + 10% FBS. Two samples were removed at 0, 24, 48, 72, 96, 120 and 144 hours after the last manipulation. Later, the samples were washed in PBS, fixed in metanol and Giemsa stained. The presence of intracellular parasites was evaluated in 500 cells ramdomly choosen under light microscope. In another set of experiments, Choachi strain parasites were pre-incubated with homologous heat inactivated immune serum or with Rhodnius prolixus salivary glands extract for 1 hour at room temperature. After washing, parasites were incubated with the cells as described previously. The results obtained demonstrated that both strains presented a similar infection rate at 24 hours of interaction (1.67±0.72% for SC-58 B1 clone and 1.90±1.14% for Choachi). Analysis in later points showed that Choachi strain parasites were not present inside of the cell after 96 hours. In contrast, for the SC-58 B1 clone cell, the infection rate was of 0.53±0.53%. For immune serum pre-incubated parasites, we have observed a higher cell infection rate when compared to the controls. Infection rate for pre-incubated parasites range from 3,17±0,54% to 0.20±0.20% at the beginning and 96 hours respectively. However, when parasites were incubated with salivary glands extract no modification in the cell infection rate was observed. The gradual decrease in the cell infection rate and absence of dividing intracellular parasites suggest that T. rangeli infects at low rate the J774G.8 murine promonocytic cells, but is unable to multiply within it

Supported by PIBIO/UFSC and CNPq.

077

CONTRIBUITION FOR THE ISOLATION, CULTIVATION AND MAINTENANCE OF THE FREE-LIVING AMEBAS.

Giazzi, J.F.; Buainain, A.; Rosa, J.A.da; Martini, A.S.; Belda Neto, F.M.; Martinez, I. & Fernandes, M.Z.T.

Departamento de Análises Clínicas e de Ciências Biológicas da Faculdade de Ciências Farmacêuticas de Araraquara UNESP Rod. Araraquara/Jaú, km 1, CEP 14.801-902.

INTRODUCTION: Free-living amebas are the most recently discovered protozoa there can produce harmful and lethal efects for the human life.They can invade the Central Nervous System and others organs, occasioning death or permanent incapacity (MARTINEZ, 1985).

MATERIAL AND METHODS: technics of direct examinations and culture are utilized, employing the Pavlova's Medium, Pavlova gel, solid Pavlova, Brewer and Diamond, in tubes and microculture between lamina and cover sleep ( in humidity chamber) samples of waters, dusts, sands and nasofaringean secretions of various origen were examined.

RESULTS: The obtained results were the meeting of free-living amebas in the analysed samples. In the one hundred and fifteen samples of investigated waters, amebas of the Acanthamoba genus were revealed in fourteen samples, and amebas of the Naegleria genus, in nineteen samples. In twenty three samples of analysed dusts, twenty positive occurrences of Acanthamoeba sp. amebas and nive occurrences of Negleria sp. amebas were found. We found for samples of both amebas of Acanthamoeba genus and amebas of Naegleria genus in the five samples of sands we analysed. Finaly, in eight samples of nasal secretions examined, amebas of Acanthamoeba genus were found in four of them.

CONCLUSION: The investigation is concluded alerting to the high diffusion of the reported protozoa in the nature and detaching the role represented by the Culture Mediuns used in the experiment, mainly by the performance of the Pavlova gel Culture Medium, that showed itself excellent to the isolation, development and maintenance of strains of the free-living amebas.

078

EVALUATION OF TOTAL PROTEINS OF TRYPANOSOMATIDS IN DIFFERENT PHASES OF GROWTH BY SDS-PAGE.

Rocha, K. M. , Yang, A. V. , Gonçalves, C. C., Okamura, H., Costacurta, R. A., Oliveira, F. L. de, Tano, M. S., Reiche, E. M.V., Itow Jankevicius, S & Jankevicius, J. V..

Núcleo Interdisciplinar de Pesquisa, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Caixa Postal 6001, Londrina, 86051-900, PR, Brazil.

The family TRYPANOSOMATIDAE includes more than 300 species of monoflagellate protozoa parasites of animals and plants. The family comprises 10 genera. The genera Leishmania and Trypanosoma are responsible for a broad spectrum of human and animal diseases. The genus Phytomonas is related to various plant diseases, and the genera Herpetomonas, Leptomonas and Crithidia are parasites of insects. Three strains of Trypanosomatids were studied: Phytomonas serpens 9T (Jankevicius et al,1987), Herpetomonas macgheei 163Ma (Itow Jankevicius et al,1993) and 415Ga (Londrina, Pr, 1990), isolated from tomatoes, corn and salivary glands of a phytophagous hemipteran, respectively. These trypanosomatids were cloned and maintained in complex liquid media ( GYPMI - Itow Jankevicius et al, 1993 ). The optimum growth conditions were determined as pH 6.5 - 7.0 at 28^O C and the generation time around 14 - 16 hours. The bulk cells were obtained in the lag, log and stationary phases, and for each phase was prepared an antigenic suspension ( Maizels et al, 1991 ). Protein concentration was quantified (Bradford 1970 ) and electrophoresis ( SDS - PAGE ) was carried out such as in Laemmli, 1970. Carbonic anidrase, 29 kDa, egg albumin, 45 kDa, and bovine albumin, 66 kDa, were used as M.W. markers. Protein concentration at different growth phases was studied by densitometry, after Coomassie Brilliant Blue G250 staining. The preliminary results indicated higher concentrations of proteins of apparent molecular weight: 18.5 kDa, 22.0 kDa and 37.0 kDa in the log phase when compared with the stationary phase of the 163 Ma strain; the proteins of apparent molecular weight 16.1 kDa and 26.4 kDa of the 9T strain were in higher concentration in lag phase, while the protein of apparent molecular weight 37.7 kDa were found only in the lag phase.

Financial support: CPG-UEL, CAPES and CNPq.

079

STANDARDIZATION OF A CRYOPRESERVATION TECHNIQUE TO FACILITATE THE CHARACTERIZATION OF LEISHMANIA PARASITES

Pereira, T.R; Grogl, M; Pereira; & Pereira, E.R.R.

USA Medical Research Unit, Walter Reed Institute of Research & Instituto de Biologia do Exército, Rio de Janeiro, Brasil.

The parasitological diagnosis of cutaneous leishmaniasis requires the visualization of the parasite in/from the clinical sample. Culturing clinical samples and characterization of the isolate by cellulose acetate electrophoresis (iso-enzymes) requires to work under sterile conditions. These conditions are generally not found in most field laboratories. It is therefore of pratiacal importance to have an easy and fast technique to freeze and transport clinical samples (tissues) from the field to a cetral laboratory. Different procedures for freezing and storing biopsies were evalueted to insure the viability of the amastigotes. A biopsy from the nose of a L. mexicana infected hamster, was divided in 24 pieces, and cryopreserved at -70^oC or freeze for 24 horas at -70^oC and them placed in liquid nitrogen. Previously to cryopreservation the tissue samples were kept for 30 minutes in PBS plus 3% Streptomycin/Penicillin (SSP) solution in a Petri dish. Biopsy samples were cryopreserved in SSP solution plus 10% dimethylsulfoxide (DMSO). Samples cryopreserved by these two methods were thawed on days 7, 14 and 21 and divided in 2 fragments. Fragments were cultured in either Schneider's Drosophila media 23% Fetal Bovine Serum 1% SSP or in NNN media. Cultures were kept at 25^oC. Amastigotes were viable by both cryopreservation methods for the total of the 21 days tested. Thus suggesting that these methods an be used to cryopreserve human biopsies permitting their transportation to a central laboratory.

080

APOPTOSIS IN A CANINE MODEL OF ACUTE CHAGASIC MYOCARDITIS

Zhang, J¹, Andrade, Z.A ², Andrade, S.G.² ,Takeda, K¹, Sadigursky, M³, Ferrans, V.J.¹

1 - Pathology Section, National Heart, Blood & Lung Institute, NIH, Bldg10/2N240 , Bethesda, MD,20892.

2 - Centro de Pesquisas Gonçalo Moniz, FIOCRUZ/ Rua Valdemar Falcão, 121, 40295-001Salvador, Ba.

3- Dep. Patologia. Faculdade de Medicina, UFBA

Histologic, ultrastructural and nick and labeling (incorporation of fluorescein-labeled deoxynucleotides into DNA by terminal deoxynucleotidyl transferase) studies were made to evaluate the occurrence of apoptosis in the hearts of dogs with acute myocarditis due to infection with Trypanosoma cruzi. The expression of Bcl-2 , Bax and Fas in various cell types and in T. cruzi was examined by immunohystochemical method (Texas red-conjugated antibody). Nuclei were stained with DAPI and the preparation were examined by confocal laser scanning microscopy. Apoptosis was found in : 1) some of the cardiac myocytes and a few endothelial cells of capillaries and venules; 2) immune effector cells, including macrophages, interstitial dendritic cells (antigen presenting cells) and granular and agranular lymphocytes, and 3) intra and extracellular forms of T. cruzi. Electron microscopic study confirmed these findings by disclosing changes of nuclear and cytoplasmic condensation, consistent with apoptosis, in all of these cell types. The apoptosis in myocytes and endothelial cells was associated with increased reactivity for Fas and Bax and decreased reactivity for Bcl-2. It affected cells that were not infected by T. cruzi and probably was caused by the release of toxic mediators of inflammation. The apoptosis of immune effector cells could be related either to subsidence of inflammation or to modulation (and even failure) of the immune response. The finding of apoptosis in T. cruzi confirms other studies (Amiesen et al.) showing that this phenomenon occurs during the differentiation of epimastigotes into trypomastigotes of T. cruzi in vitro . Thus, apoptosis constitutes and important and multifactorial event in the pathogenesis of acute chagasic myocarditis. Pharmacologic modification of this apoptosis may be of therapeutic importance in Chagas' disease.

THE MEGACOLON, AN EXPERIMENTAL EVIDENCE IN HAMSTER INFECTED WITH TRYPANOSOMA CRUZI

Ramirez LE., Lages-Silva E., Marfrin A., Oliveira DI., Matos A., Santos MC., Messias A., Pereira G., Chapadeiro E.

Disciplinas de Parasitologia , Patologia e Imunologia, FMTM, Uberaba, MG

Since 1991 we have been studying the hamster as an experimental model in Chagas' disease. It was observed since then that this animal presents lesions very similar to those found in humans , both in heart and in the digestive tract. We demonstrated (Chapadeiro e cols. 1996) the development of megas, mainly in stomach, colon and cecum by macroscopic, microscopic and radiologic analysis. Nevertheless, the radiologic analysis didn't permit the accurate discrimination of different portions of bowel, leading to results with little definitions. In this data we utilized 40 young hamsters, non-isogenic, twenty (20) of then infected with "Vicentina" strain of T. cruzi and twenty controls. These animals were individually separated in cages and fed with ration plus barium as contrast. During 15 days, feces of these animals were collected, stored at room temperature and examined radiologically, in order to study the intestinal transit through time of the contrast elimination.

The results demonstrated, through three observations done from the 6^th month of inoculation, that as the infection evolves there is a tendency of the animal to develop megas: 1^st lecture (15.6%), 2^nd (11.5 %), 3^rd (36.8%). It's important to notice that in three animals there were a ponderal loss of rapid evolution and also obstipation for over eight days. After the animals were sacrificed and necropsied there were observed the development of megacolon and a kind of "baritoma" in one of them. Histologically the cecum and the junction of pre-stomach and stomach were the regions where the great number of lesions characterized by inflammatory process with infiltrate of mononuclear cells, mainly macrophages and plasmocyte was observed.

We concluded that the hamster chronically infected with T. cruzi shows a retard on the intestinal transit suggesting a disturb of motility of bowel due to alterations of the autonomic intramural nervous system. This is the only known experimental model which shows characteristics alterations of enteromegalia.

Supported by: FAPEMIG

082

VASOACTIVE INTESTINAL POLYPEPTIDE AND SUBSTANCE P IMMUNOREACTIVE NERVE FIBERS IN THE MOUSE COLON , IN THE CHRONIC PHASE OF T. CRUZI INFECTION.

De Souza, R.R. ; Maifrino, L.B.M. ; De Souza, H.M.R. ; Liberti, E.A.

Departamento de Anatomia - ICB III - Universidade de São Paulo , Instituto Dante Pazzanese de Cardiologia .

Several immunohistochemical studies have provided evidence that a number of the neuropeptides are found in nerve fibers in the myenteric plexus of the gut wall, most notably vasoactive intestinal polypeptide (VIP) and substance P (SP). Furthermore, experimental evidence seems to , indicate that these peptides could be related to the granular vesicles observed in the nerve endings of the myenteric plexus. In the present it is assumed that these peptides function as neurotransmitters. It is Known that during the acute phase of Chagas'disease, there is a decrease in the neurosecretory vesicular component of myenteric plexus, as well as a significant reduction in SP and VIP. However , little is known about these types of neurotransmitters during the chronic phase of the disease. In the present study immunohistochemical and ultrastructural methods have been to examine the distribution of VIP and SP immunoreactive fibers in the mouse colon infected with T. cruzi , in the chronic phase of the infection. A number of varicose VIP - and SP - positive nerve fibers , running parallel to the muscle bundles, were seen in the control animals. In the ganglia of the myenteric plexus numerous VIP - and SP - positive fibers were common and often surrounded the neuronal cell bodies , which were always immunonegative. In contrast , in the chagasic animals , both the density and staining intensity , were always lower in these locations than in the corresponding sites in the control group. The number of granular vesicles per axonal area in the myenteric plexus of the colon was smaller in mice inoculated with T. cruzi than in control animals.

083

MORPHOMETRIC INVESTIGATIONS OF THE COLON MUCOSA AND MUSCLE LAYER IN CHRONIC T. CRUZI INFECTED MICE.

Maifrino,L.B.M. ; Liberti, E.A . and De Souza, R.R.

Instituto Dante Pazzanese de Cardiologia , Departamento de Anatomia - ICB III -Universidade de São Paulo .

It was demonstrated that after denervation of the myenteric plexus induced by T. cruzi infection there was hyperplasia of the rat colon epithelium and thickening of the mucosa of the human sigmoid colon. Furthermore, it was also observed muscle hypertrophy during the initial stages of visceral dilatation in Chagas'disease. The objective of the present investigation was to study the morphometry of the mucosa and of the muscle layer of the mouse colon in the chronic phase of T. cruzi infection. Seven young male Swiss mice were inoculated with the Y strain of T. cruzi. Two months after inoculation, the animals were sacrificed and the distal colon was collected for morphometric measurements of the volume of the muscle layer, the number of the neurons in the myenteric plexus , and the area of the mucosa. There was an increase in the volume of the muscle layer , a significant denervation and an increase in the area of the mucosa in the group inoculated with T. cruzi. These data suggest that denervation of the myenteric plexus induce hypertrophy of the muscle layer and of the mucosa of the colon in T. cruzi- infected animals. It is possible that these alterations may account for the abnormal colonic motility, secretion and absorption observed in the chronic phase of Chagas'disease.

084

STRUCTURAL ANALYSIS OF TRYPANOSOMA CRUZI AFTER INTERACTION WITH CELLULAR SUBFRACTIONS FROM HUMAN PLACENTA.

Asteggiano C., Sartori MJ., Lin S., Frank F., Fabro S.P.de and Fretes R.E.

IIa. Cátedra de Histología y Embriología. Fac. Cs. Médicas. Universidad Nacional de Córdoba. Agencia Postal Nº4, (5000) Córdoba, Argentina. Suppported by CONICOR and SECyT (U.N.C.).

The purpose of the present work was to analyse the survival and ultrastructural changes of Trypanosoma cruzi after their interaction with cellular subfractions of human placenta. Subsceptible to T. cruzi infection VERO cells were used as control. Subcellular fractions from normal placentas at term and VERO cells were obtained according to Corash and Gross (1973). Tulahuen strain of trypomastigotes were isolated from mouse blood following De Titto et al (1986). The interaction of 285 mgr of protein (Lowry 1965) from different cellular subfractions (Homogenate, Nuclear, Mitocondrial, Lysosomal and Supernatant) with 10⁶ parasites for one hour at 37ºC in M-199 culture medium was performed. After the interaction parasites were processed both for viability by a dye exclusion test in a Neubauer chamber and for electron microscopy. Routine technique and cytochemistry for acid phosphatase, alkaline phosphatase and rutenium red tincion were done. It was found that 79.6±10.5% and 77.9±15% of trypomastigotes were dead after their interaction with Lysosomal and Supernatant fractions respectively. These survival values were lower in comparison with other fractions and control (parasites without placental proteins) (p<0.05). Parasites were not affected in their viability after interaction with none of the cellular subfractions of susceptible VERO cells. Ultrastructural studies showed different degrees of parasites damages, presumely as result of lysosomal components' action. The protozoo cellular body was greatly vacuolized, plasmatic membranes were altered and in some samples an increase of total parasitic destruction could be observed. These modifications were accompained with positive acid phosphatase reaction in parasites cytoplasm. Alkaline phosphatase reactions were observed in syncytial membranes and intracytoplasmatic membranes of the trypomastigotes. With rutenium red the loose of parasites plasmatic membranes glycoproteins could be observed, with different degrees of alteration. We concluded that there are selective trypanocide agents in Lysosomal and Supernatant fractions of normal placentas which are not present or exist in a lower concentration in other cellular subfractions and VERO cells. This suggests that placental lysosomal components could selectively alter the structure of typomastigotes and the increase of lysosomes in placentas from chagasic women (Fretes and Fabro 1995) could prevent or limit the transplacental infection, explaining, at least in part, the low incidence of congenital Chagas' disease.

085

REFLEX CONTROL OF HEART RATE AND ARTERIAL PRESSURE IN EXPERIMENTAL CHRONIC CHAGAS' DISEASE IN RATS.

Dias da Silva¹, VJ; Fernandes, PS¹; Ramirez, LE¹; Teixeira, VPA¹; Chapadeiro, E¹; Salgado, HC²

¹Department of Biological Science, FMTM, Uberaba, MG, Brazil, ²Department of Physiology, FMRP/USP, Ribeirão Preto, SP, Brazil

Human chronic Chagas' heart disease is characterized by a progressive fibrosing myocarditis usually associated with peripheral autonomic neuropathy of varying extent. Lately, the rat experimental model of this pathology has obtained special attention. The aim of this study was to evaluate the baro, cardio-pulmonary (Bezold-Jarisch) and chemoreceptor reflex control of HR and AP of rats chronically infected with the "Y" strain of Trypanosoma cruzi. Male Wistar rats (n=12)were inoculated intraperitoneally with 2x10⁶T. cruzi blood forms. On the 10^th day, the infection was confirmed by positive parasitemia. Control rats (n=10) were inoculated with blood without parasites. After 6 month, the right femoral artery and vein were cannulated, under anaesthesia, for recording AP and HR and drug administration, respectively. After 24 h, the animals had their basal AP and HR recorded during 30 minutes. Then, the baroreflex control of HR was investigated by iv administration of phenylephrine (1-16mg/Kg) and sodium nitroprusside (1-64mg/Kg). Changes in mean AP(MAP) and HR induced by the drugs were used to calculate the baroreflex sensitivity by linear regression. On following day, the Bezold-Jarisch and chemoreceptor reflexes were studied, respectively, by iv administration of phenylbiguanide (PBG, 1-10mg/Kg) and potassium cyanide (KCN, 40-320mg/Kg). Basal values (mean±SEM) of MAP and HR did not differ between groups (114±5mmHg and 368±19bpm vs. 106±4mmHg and 382±19bpm in control rats). Sensitivities (bpm/mmHg) of reflex tachycardia and reflex bradycardia (-3,3±0,9 and -1,9±0,3 and -2,7±0,3 and -2,1±0,3 in control rats, respectively) and dose-response curves to PBG and KCN did not differ between groups. These data indicate that, in chronic chagasic rats, the baro, cardio-pulmonary and chemoreceptor reflex control of HR and MAP are well preserved.

Supported by: CAPES, FAPESP, FAPEMIG

086

EFFECT OF CHAGAS' DISEASE ON TWO-KIDNEY-ONE-CLIP HYPERTENSION IN RATS.

Fernandes, PS; Macedo, CFC; Souza, JC; Ramirez, LE; Dias da Silva, VJ & Chapadeiro, E.

Department of Biological Sciences, School of Medicine of Triangulo Mineiro, Uberaba, MG, Brazil.

In literature, there is scant information in relation to arterial blood pressure and Chagas' disease. Palmero et al. (Am Heart J., 97: 38-42, 1979) described that arterial blood pressure of chagasic men and women was significantly lower than of the general population. Otherwise, the interaction between Chagas' disease and arterial hypertension is little known. The aim of this study was to investigate the effect of Trypanosoma cruzi inoculation on arterial blood pressure of two-kidney-one-clip (2K1C) hypertensive rats. Twenty three male Wistar rats were submitted to implantation of a silver clip on left renal artery to induce renovascular hypertension. After 04 weeks, fifteen hypertensive rats (Chagasic group) were inoculated intraperitoneally with 2 x 10⁶ "Y" strain Trypanosoma cruzi blood forms. The infection was confirmed by positive parasitemia. Eight hypertensive rats (Control group) were inoculated with blood without parasites. Then, the animals were observed during 04 weeks after inoculation, corresponding to acute phasis of Chagas' disease in rats. During all experimental period (08 weeks), systolic arterial pressure (SAP) and heart rate (HR) were monitored weekly using a indirect blood pressure meter (LE-5000, Letica SA, Barcelona, Spain). In the end, the rats were sacrified and the hearts were weighed and submitted to pathological studies. The final corporal weight was similar between groups (313±11 versus 308±15 g. in control group). SAP was similar before (187±10 versus 194±13 mmHg in control group) and after 04 weeks of inoculation (211±9 versus 232±14 mmHg in control group). HR was similar before inoculation (371±15 versus 352±12 bpm in control group), but during the 1^st, 2^nd , 3^rd and 4^th weeks of acute phasis there was a tachycardic response in chagasic rats (400±10*, 409±12*, 376±11* and 378±9 bpm versus 322±18, 355±7, 338±10 and 382±18 in control group, * p < 0,05). The relative heart weight was not different between groups (4,78±0,44 versus 4,72±0,53 mg/g in control rats). These data indicate that arterial blood pressure and heart weight of 2K1C hypertensive rats were not changed during acute phasis of Chagas' disease in rats. The tachycardic response was similar that observed in normotensive rats (Gottberg et al., Trans.R.Soc.Trop.Med.Hyg., 82(6):851, 1988). The effect of the chronic Chagas' disease on 2K1C hypertension in rats will be investigated.

Supported by: FAPEMIG

087

SMOOTH MUSCLE SYMPATHETIC INNERVATION OF THE MALE GENITAL ORGANS DURING THE INFECTION WITH DIFFERENT Trypanosoma cruzi ISOLATES, IN RATS

Franco, DJ, Garcia, CMMG, Teixeira Jr, AL, Camargos, ERS, Chiari, E, Machado CRS.

Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Caixa Postal 486, Belo Horizonte, 31270-901, MG, Brasil.

Previous studies have shown no alteration in the sympathetic innervation of the ductus deferens of rats inoculated with 300,000 trypomastigotes of Y strain. In the present paper, the noradrenergic innervation of the rat prostate, seminal vesicle and ductus deferens was studied by a glyoxylic acid-induced histofluorescence method for catecholamines at the end of the acute infection with different strains or clones of T. cruzi (10,000 trypomastigotes ip in rats aged 27-29 days). The parasitemic curve presented low values (maximum of 600 tripomastigotes/5 ml of blood) in all animals inoculated with the Col 1.7G2 clone (from Colombian strain), the mortality being less than 2 %. Infected and control animals exhibited the same pattern of innervation in all organs studied and no inflammatory processes or amastigote nests were observed. With the CL-Brener clone, the animals comprised four groups according to the parasitemic curve: low parasitemia (peak less than 1,000 trypomastigotes/5 ml); moderate parasitemia (less than 2,500); high parasitemia (up to 7,000); very high parasitemia (above 10,000). Most animals with moderate and all animals presenting high or very high parasitemia died from day 23 to 29 of infection. At the end of the acute phase, only the prostate showed moderate to severe noradrenergic denervation in 67% of the infected animals, along with moderate to severe diffuse inflammatory processes. The infection with ABC strain was characterized by three parasitemic patterns: low parasitemia (peak up to 1,000), moderate (till 8,000) and high (above 15,000). Regardless the parasitemic pattern all animals died around day 20 of infection. At day 20 of infection, all genital organs showed moderate to severe rarefaction of the noradrenergic nerve terminals. In the ductus deferens the rarefaction was restricted to the peripheral third. The histological studies showed diffuse inflammatory processes in the prostate and seminal vesicle. However, in the ductus deferens these processes were found mainly in the peripheral third. The smooth muscle of the seminal vesicle and ductus deferens seems to be spared during the acute phase of T. cruzi infection with Col 1.7G2 and CL-Brener clones, even when the parasitemia reaches very high levels (CL-Brener). In contrast, the ABC strain was able to cause rarefaction of the nerve terminals in all organs studied, regardless the parasitemic pattern. However, even in the ABC infection the middle and inner thirds of the ductus deferens smooth muscle layer remain unaltered.

CNPq, PRONEX-1996, CAPES

088

ACUTE TRYPANOSOMA CRUZI INFECTION IN RATS: THE PARASITEMIA, HEART SYMPATHETIC DENERVATION AND INFLAMMATORY PROCESSES DEPENDS ON THE PARASITE ISOLATES

Garcia, CMMG, Franco, DL, Dutra, AP, Teixeira Jr, AL, Camargos, ERS, Chiari, E & Machado, CRS.

Instituto de Ciências Biológicas, Caixa Postal 486, Belo Horizonte, 31 270.901, Minas Gerais, Brasil.

It is well established that young rats inoculated with 300,000 trypomastigotes of the Y strain present severe myocarditis and complete sympathetic denervation at the end of the acute phase (20 days). Recently, different patterns of parasitemia were found in young rats inoculated with 10,000 trypomastigotes of the Y strain. The highest levels of parasitemia were related to severe sympathetic denervation (Dutra et al., 1996 - Braz. J. Morphol. Sci., 13:128-129). In the present study, we investigate the involvement of the heart sympathetic innervation during the acute phase of the infection with different T. cruzi isolates, aiming at comparing the denervation with the parasitemia. All rats were inoculated intraperitoneally with 10,000 trypomastigotes of COL 1.7G2 clone, or CL-BRENER clone, or ABC strain at the age of 27-29 days. The infection with the COL 1.7G2 clone caused low parasitemia (less than 600 parasites/5 ml of blood along 42 days). Moderate denervation occurred in atrial tissue of 33% of the infected animals at the end of the acute phase (30-42 days of infection). The parasitemia during the infection with CL-Brener clone could be classed as low, moderate, high, and very high (as described by Franco et al., this Meeting). Twelve out of thirteen infected animals killed at days 24-29 of infection presented moderate (one) to severe (eleven) sympathetic denervation in atrial tissue, regardless the parasitemic curve. The infection with the ABC strain (parasitemia in Franco et al., this Meeting) killed all the animals around day 20 and caused severe to complete denervation of the atrial tissue, even in the rats with low parasitemia.

In animals sacrificed at days 12-15 (seven animals) and 19 (nine) of T. cruzi infection with Y strain (10,000 trypomastigotes, ip), the atrial inflammatory processes were quantified and compared with the parasitemia and the heart sympathetic denervation. Our results showed no correlation between parasitemic curve and atrial inflammatory processes. It seems however that inflammation relates somehow to the degree of sympathetic denervation.

CNPq, PRONEX-1996, CAPES

089

CALCIUM CURRENT ALTERATIONS IN ISOLATED RABBIT CARDIAC MYOCYTES TREATED WITH SERA FROM CHRONIC CHAGASIC PATIENTS.

Barcellos, LC; Pedrosa RC; Campos de Carvalho, AC & Masuda MO. Instituto de Biofisica Carlos Chagas Filho. UFRJ, RJ.

Interaction between sera of chronic chagasic patients and muscarinic and b adrenergic receptors has been demonstrated by several laboratories. This interaction may be due to molecular mimicry between parasite ribosomal proteins and the second extracelular loop of G-protein coupled receptors. Recently Farias de Oliveira et.al. (Circulation, 1997), demonstrated that sera from chronic chagasic patients, can induced conduction block and decrease in heart rate in isolated rabbit hearts and this effect was abolished by atropine. Activation of muscarinic receptor by these sera should result, among other things, in a reduction in calcium currents through the cardiac myocyte's sarcolema. To test this we dissociated ventricular myocytes from rabbit hearts and analyzed them by whole cell patch clamp technique. To isolate the calcium currents we used Tyrode solution containing (mM) NaCl 150.8, KCl 2.7, MgCl 0.5, Glucose 6.0, HEPES 10.0, CaCl₂ 2.7 with the potassium blocker 4- Aminopyridine (2 mM), and a pipette solution containing (mM), CsCl 120. NaCl, 20, CaCl₂ 0.5, HEPES 10, TEA-Cl 20. Isoproterenol (10 ^-9 M) was added to the bath to increase the current. After control records in Tyrode we perfused the cells with the same solution containing sera from chronic chagasic patients at a dilution of 1:100 (v:v), for a period of 5 min before returning to tyrode. Our results show marked reduction in calcium currents amplitude (1221 ± 131 pA to 809±137 pA, n=4, p<0.001) during perfusion with the sera. Perfusion with normal sera did not effect the calcium current. This date are compatible with the effect of sera from chronic chagasic patients being mediated through activation of muscarinic receptors.

Financial Supports by CNPq, finep, faperj, Pronex

090

THE EFFECTS OF OVARIECTOMY AND ORCHIECTOMY ON THE HISTOPATHOLOGICAL PROFILES OF CALOMYS CALLOSUS INFECTED WITH THE "Y" STRAIN OF T.CRUZI.

Prado Jr.,J.C.^1,2, Alves, C.², Leal, M.P., M.L.² & Andrade Jr. H.F.² .1 Dept. Ciências da Saúde, Fac. de Ciências Farmaceuticas, USP, Av. do Café s/n Ribeirão Preto, 14.049-903, Brasil 2 Inst. Medicina Tropical S.Paulo.

Distinct aspects of the sex-dependent differences in the magnitude of the immune response have been studied in the wild rodent Calomys callosus. Female C.callosus are more resisitant to T.cruzi infection when compared to males. In this work we evaluate the histopathological profiles of C.callosus during the course of T.cruzi infection. Male and female C.callosus were divided into 3 groups: Operated, "Sham" (Simulated operation) and Control. One month after surgery, all animals were inoculated i.p.with 4 x 10³ blood trypomastigotes of the "Y" strain of T.cruzi. On pre-determined days 3 animals of each group were sacrificed. Fragments of heart, spleen and thymus were collected, fixed in 10% formaldehyde and included in paraffin. Histopathological sections of 7mm were stained by haematoxilin-eosin, and individual slides of each organ were analyzed with different amplifications. A direct correlation between parasitemia, hormonal status and tissue involvement was observed. Distinct behaviours can be induced by T.cruzi infected C.callosus submitted to ovariectomy or orchiectomy. Castrated females, become more susceptible, with high parasitemia levels and a more intense tissue reaction when compared to their sham and control groups. Orchiectomized animals become less susceptible with lower parasitemia levels and lesser tissue involvement. Heart sections of ovariectomized females and intact males showed an extensive inflammatory process and higher amastigote loads. Also, intact females and orchiectomized males, showed a reduced amastigote load and a less intense tissue injury. Germinative follicles of spleens were more activated in orchiectomized animals, when compared to their controls, while in ovariectomized females they appeared looser with a more intense red pulp proliferation. Thymus sections from animals of both sexes showed a higher thymocyte proliferation for ovariectomized and orchiectomized animals. The histopathological details will be presented at differente amplifications. In spite of a greater or lesser susceptibility due to surgery, T.cruzi infection did not induce C.callosus mortality.

Financed by CNPq, FAPESP and LIM 49-HC.

THE ROLE OF REINOCULATION ON THE FOLLOW UP OF CHRONIC CHAGASIC INFECTION IN HAMSTER: MACROSCOPIC AND MICROSCOPIC ASPECTS

Ramirez LE., Matos A., Lages-Silva E., Santos MC., Miziara JM., Reis JD., Pedrosa AL., Bittar PCM., Chapadeiro E.

Disciplinas de Parasitologia e Patologia, Departamento de Ciências Biológicas, FMTM, Uberaba, MG

Since now hasn't been elucidated the role of reinoculation on the course of infection by T. cruzi, specially on the development and intensity of tissular lesions. In this present data our proposal is to study, the role of reinoculation on the course of chronic chagasic infection in hamster.

One hundred (100) animals (hamster) were injected with 5 x 10 ³ tripomastigotes of the "Vicentina" strain on the following way: all the animals were infected at time zero and ten animals, among the survivors, were randomly choosen and reinoculated with the same strain, after 60 days. This procedure was repeated at each 40-50 days untie the 6^th time. During this period all the animals which presented themselves in a bad general state were sacrificed. Then animals were used as controls. Nearly one year after the first inoculation the survinging animals were also sacrificed after ether anesthesia, proceeding the collection of blood for parasitologic exams: microhematocrit (Mhto) and hemoculture (HC). During necropsys, samples of all organs were obtained for histopathologic studies.

The macroscopic results showed that from the 45 infected and sacrificed animals 17 (37.8%) didn't presented alterations. Among the resting it was observed: 11 (24.4%) with enteromegalia, 8 (17.8%) with cardiomegalia with signs of cardiac insufficiency, 9 (20%) with cardiomegalia associated to enteromegalia, 9 (20%) with biliar vesicle dilation, 5 (11.1%) with atrophy of external genital organs, 3 (6.7%) with hypertrophy of adrenal and 2 (4.4%) with anasarca. The mortality index increased from 10% to 65% from the first to the 3 ^rd inoculation, being constant from then. Among the controls no lesion was observed. A relation between the ponderal loss and the presence of megas of digestive tract, many times followed by cardiac atrophy, was observed. In those animals presenting cardiomegalia it was found pleural and ascitic liquid in variable amounts, liver and spleen congested characterizing a cardiac insufficiency. These hearts many times presented intramural thrombus. The parasite was recovered even after many inoculations through the parasitologic methods utilized: Mhto (8.6%), HC (14.3%). Attention may be done over some animals that although inoculated only once, presented so important symptoms as those receiving many inoculations. Microscopic lesions were classified as light (L), moderated (M) and serious (S) and showed the following distribution: esophagus (58.3%L and 8.3%M); stomach and junction esophagus and pre-stomach (10%L, 40%M and 30%S); small intestines (30%L); colons (60%L and 10%M); cecum (54.5%L, 9.1%M and 27%S). In heart: atrium (37.5%L, 43.8%M and 18.7%S), ventriculum (62.5%L, 25.0%M and 12.5%S). It also was observed amiloidosis in adrenal ( 87.5%), spleen (57.1%), liver (20%) and kidneys (80.0%). Nefroesclerosis was noticed in 46.6%. In control animals no lesion was found.

It was concluded that reinoculation is not a process absolutely necessary to develop lesions although it can be important to improve its frequency.

Supported by: FAPEMIG

092

TRIATOMINAE SALIVA CONTRIBUTES TO PATHOGENICITY OF TRYPANOSOMA CRUZI.

Sherlock, I. A, Sadigursky, M.

Centro de Pesquisas Gonçalo Moniz / FIOCRUZ e Faculdade de Medicina da UFBa. - Salvador, Bahia.

During triatominae blood feed, the vertebrate tissue is damaged resulting in platelet activation and local vascular constriction. In few seconds this reaction is refrain by platelet deagregation factors and vasodilator produced by the insect. This process that facilitates the insect feed act also in the vector/T.cruzi/host interaction adapting the protozoa maintenance and transmission. In the present work it was investigate the action of products injected by the insect associated to T. cruzi in the pathogenesis of Chagas' disease. Six outbred Swiss mice infected with 10² trypomastigote forms of T. cruzi (Y strain) injected intraperitoneously constituted group I, eight Swiss mice infected intraperitoneously with 10² trypomastigote forms associated with product of one T. infestans salivary gland constituted group II and six mice inoculated with product of one T. infestans salivary gland constituted group III ( control). Parasitemia was determined each four days and after 60 days post inoculation all the mice of the three groups were sacrificed and segments of the principal organs were fixed in 10% formalin and processed for histopathological study. All the mice of group I, presented moderate to diffuse myocarditis (4/2) while mice of group II infected with T.cruzi associated to products of salivary gland 4 presented moderate, diffuse myocarditis and 4 presented moderate, focal myocarditis. Control animals did not presented myocarditis. The data presented suggest that products of T. infestans salivary gland contribute to modulation of the inflammatory process of heart in experimental Chagas' disease.

093

INTERACTION OF TRYPANOSOMA RANGELI WITH MIDGUT CELLS OF RHODNIUS PROLIXUS TREATED WITH LECTINS

Oliveira, M.A.* & De Souza, W.*^,**

*Universidade Estadual do Norte Fluminense, Centro de Biociências e Biotecnologia, Lab. de Biologia Celular e Tecidual, Campos dos Goytacazes-RJ, Av. Alberto Lamego, 2000, 28015-620. **Universidade Federal do Rio de Janeiro, Inst. de Biofísica, Lab. Ultraestrutura Hertha Meyer, Ilha do Fundão, Rio de Janeiro, 21949-900, Brazil.

Trypanosoma rangeli is an hemoflagellate protozoan that parasitizes a wide range of mammals and triatomine bugs. Its distribution comprise Central and South America, and its most important vector is the triatomine Rhodnius prolixus, the same vector of T. cruzi. This parasite is pathogenic for the invertebrate host, naturally or experimentally infected. The infection of the vector happens during blood meal and its transmission occurs when the infected bugs bite the mammals inoculating parasite together with saliva during the blood meal. Previous studies have shown that carbohydrate containing macromolecules are involved in the process of parasite-host interaction. In the present study lectins were used to analyse the involvement of carbohydrate residues exposed on the surface of midgut cells of R.prolixus on the interaction with T. rangeli. The initial 5mm of midguts were dissected and washed 3 times in 0,1M PBS pH 7.2. The intestines were fixed in 1% formaldehyde diluted in PBS pH 7.2 for 10 min, washed 3 times in the same buffer and sequentially incubated with 50mM of NH₄Cl for 90 min. and PBS/ 1% BSA for 20 min. to block unspecific binding sites. The following lectins, diluted to a final concentration of 10mg/ml in PBS/ 1% BSA, were tested: HPA, PNA and ConA. The tissue fragments were incubated for 90 min. at room temperature, after which they were washed 3 times. The parasites (Choachi strain) were cultivated in LIT medium with 10% BSA and 20% fetal calf serum, washed 3 times in PBS pH 7.2 and diluted to a final concentration of 10⁶ parasites/ml. Parasites and intestinal fragments (control and previously incubated with lectins) were allowed to interact for 90 min, then washed in PBS and disrupted with a teflon style in 200ml of PBS. The number of parasites adhered to the midgut was determined counting the parasites remaining in suspention in a hemocytometer chamber. The results suggest that HPA and PNA inhibit T. rangeli adhesion (54% and 61%, respectively) and ConA enhanced the adhesion in 45%. These results suggest that more than one carbohydrate exposed on the surface of midgut are involved in the process of adhesion of T. rangeli to the intestine of R.prolixus.

Supported by: PRONEX, CNPq, FINEP, FENORTE.

094

ULTRASTRUCTURAL STUDY OF THE SALIVARY GLANDS OF RHODNIUS DOMESTICUS INFECTED WITH TRYPANOSOMA RANGELI.

Izabel S. Rodrigues, Mário Steindel¹ & Maurilio J. Soares.

Departamento de Ultra-estrutura e Biologia Celular. Instituto Oswaldo Cruz / FIOCRUZ, Av. Brasil 4365 Manguinhos, 21045-900 Rio de Janeiro, RJ. 1- Departamento de Microbiologia e Parasitologia, Centro de Ciências Biológicas, UFSC, Caixa Postal 475. 88040-900 Florianópolis, SC.

Rhodnius domesticus is the main invertebrate host of Trypanosoma rangeli in Santa Catarina, Brazil. The parasites invade the salivary glands (SG), where the metacyclic forms are found. Therefore, data on the morphology and physiology of these organs may help in understanding the host-parasite relationship. Thus, in this study we analyze the ultrastructure of the SG of normal and T. rangeli-infected R. domesticus by scanning (SEM) and transmission (TEM) electron microscopy. Adult insects of both sexes were used. The SG were obtained 20 days after feeding, or else 20/28 days after infection with T. rangeli.

By SEM, the SG appear as 1 mm long unilobular structures. A excretion channel arises at the medial/sub-terminal region, with a round structure at its basis. Bundles of muscle cells envelop the glands, running transversally to the major axis. TEM showed that the SG are surrounded by a basement membrane, 0.3-0.7 mm thick, where smooth muscle cells as well as traqueal cells are immersed. In some regions the basement membrane appears as a multilayered structure. These layers split apart to envelop the muscle and traqueal cells, forming an outer and an inner basal membranes, which eventually refuse again. In infected glands several parasites are found outside or inside these layers.

The SG is formed by a monolayer of flatenned epithelial cells, about 18 mm high, interconnected at their lateral membranes by interdigitations and scalariform junctions. Each cell contains usually one elongated nucleus, located at its apical portion. The cell cytoplasm contains abundant mitochondria and rough endoplasmic reticulum profiles. Few small Golgi complexes are scattered throughout the cell. The basal portion of the gland cell is usually devoid of organelles. Microvilli are present at the cell plasma membrane directed towards the gland lumen. In infected glands, parasites (mostly epimastigotes forms) are found attached to the microvilli by their flagella. Despite the high number of parasites inside and outside the SG, parasites inside the gland cells were scarce. The few parasite profiles found were inside tight vacuoles. Such data indicate that the parasites reach the gland lumen by transversing the gland cell cytoplasm. Infected glands showed no morphological alterations.

This work has been supported by CNPq, PADCT/CNPq and FIOCRUZ.

095

MANNOSE RECEPTOR EXPRESSION ON PRIMARY CULTURE OF SKIN FIBROBLASTS INFECTED BY LEISHMANIA AMAZONENSIS PROMASTIGOTES FORMS.

Côrte-Real, S., Soeiro, M.N.C., Moreira, F.A., Oliveira, G. & Meirelles, M.N.L.

Departamento de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz - FIOCRUZ, RJ.

In man, trypanosomatids of the genus Leishmania reside and multiply in its amastigote forms within mononuclear phagocytes. The parasites responsible for the different types of leishmaniasis evolved the strategy of evading the host immune system by invading monunuclear phagocyte system by receptor-mediated endocytosis. Previous studies have shown the participation of fibroblasts in the initial steps of the cutaneous leishmaniasis both in vivo and in vitro. As the Leishmania surface is particularly rich in carbohydrates, mainly mannose residues, the question arises whether the organism is recognized by a specific receptor on the fibroblasts surface, which could mediate parasite uptake. The aim of the present study was to characterize the presence and the expression of mannose receptors in primary cultures of mice embryos skin fibroblasts (SF) before and after their infection by L. amazonensis promastigote forms. SF cultures were obtained by skin dissociation using 1 mg/ml Collagenase for 1h/37ºC. Promastigotes were used to infect SF for 24 and 48h/37ºC in PBS buffer at a parasite:host cell ratio of 10:1. For detection of mannose receptors, a neoglycoprotein specific for mannosyl residues was applied (Man-BSA-PICT) employing two different protocols: 50mg/ml neoglycoprotein was added to the cultures before or after 2% PFA fixation. Peritoneal macrophages were used as controls. Mannosyl residues at the parasite and SF cell surface was detected by 100mg/ml Concanavalin-A (ConA-FITC) for 1h/37ºC after 2% PFA fixation. Our results revealed an intense mannosyl residues distribution at the parasite and SF surfaces being the intracellular parasites more strongly labeled than the host cell. No significant difference was noticed between normal and infected SF cultures. SF incubated at 4 ºC with Man-BSA displayed only a faint labeling. On the other hand, previously fixed SF showed that Man-BSA was intensively bound in non-infected SF while after the parasite infection a significant decrease in the labeling was observed. Comparing to the signal displayed at macrophage and SF cell surfaces we found that macrophages expressed less Man-BSA binding. The addition of 200mM D-mannose blocked the neoglycoprotein labeling. Literature data related to the modulation of mannose recpetor during Mf-Leishmania interaction are controversial. Our results showed a down modulation of the MR during 24-48 h interaction of primary fibroblast cultures and L. amazonensis promastigotes.

Supported by CNPq, PAPES/FIOCRUZ.

096

IRON-TRANSFERRIN BINDING ON LEISHMANIA AMAZONENSIS AMASTIGOTE SURFACE AND ITS ROLE IN INFECTION

Borges, V. M., Vannier-Santos, M. A. & De Souza, W.

Lab. Ultraestrutura Celular Hertha Meyer, Programa de Biologia Celular e Parasitologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Cidade Universitária, 21949-900, Rio de Janeiro-RJ, Brasil.

The availability of iron plays critical roles in host-parasite interplay. This metal is essential for the establishment and progress of infection by several pathogenic microorganisms (Weinberg and Weinberg, Curr. Op. Infect. Dis 8:164, 1995). We have previously observed that Leishmania amazonensis amastigotes can exploit and subvert the host cell endocytic system for iron acquisition, altering the transferrin (Tf) recycling pathway in infected macrophages (Borges et al., submitted). This study has now been further extended to determine the iron involvement on the intracellular replication of L. amazonensis. After interaction of parasites with murine peritoneal macrophages for two hours, the association indexes (AI, % infected mF x parasite/mF) were obtained 7 days post-infection. Infected macrophages treated with deferoxamine (DFO), a potent chelator of iron, reduced the rate of amastigote proliferation in a dose-dependent fashion. Addition of Fe-Tf, but not apo-Tf (iron-free Tf), increased the AI by about 50%. This result was partially reverted by combination Fe-Tf/DFO, indicating that DFO may restrict iron availability for the pathogen replication. To approach the Tf-binding activity on axenic amastigote surface, ethidium bromide (EB) was used as a quenching agent in fluorescence assay. Tf-FITC staining was diffuse at 4ºC and punctate at 31ºC and only the former was sensitive to EB, These date suggest a major role for Fe-Tf as an iron source for L. amazonensis amastigote during infection and that its endocytosis by parasite is a temperature-dependent process.

Supported by PRONEX, FAPERJ, CNPq, CAPES, FINEP and CEPG-UFRJ.

097

OUTGROWTH OF LEISHMANIA AMAZONENSIS INFECTION IN THE SANDFLY LUTZOMYIA MIGONEI: A LABORATORY STUDY.

NIEVES, E. & PIMENTA, P. F. P*

Centro de Pesquisas René Rachou- CPqRR; Fundação Oswaldo Cruz - FIOCRUZ, P. O. Box 1743. Av.Augusto Lima, 1715, Belo Horizonte - MG, CEP 30000, Brazil. Fax (031) 295-3115 E-mail : pimenta@netra.cpqrr.fiocruz.br

The complete life cycle of Leishmania spp. within the gut of potential sandfly hosts have been little studied. The present study examine the host-parasite relationships between the L. amazonensis and the sandfly Lutzomyia migonei. One of the objectives, is to evaluate the insect vector potential. We intend to settle a model for future investigations of the parasite interaction with both susceptible and resistant sandflies. In this study, L. migonei of closed laboratory colony were infected with L. amazonensis amastigotes. The parasites were isolated from a hamsters lesion and mixed with mouse blood in a concentration of 1.5 x 10 ⁷ amastigotes/ml. The sandflies were allowed to feed in a feeding-device. Engorged females were separated and maintained at 25º C, 80% RH (room humidity) and provided 50% sucrose solution ad libitum. The 7 day-old L. migonei females showed a preference to feed in a membrane feeding-device, thorough a chick skin inside a pot with with 2 cm lenght, at 25ºC, 90% RH. The sandflies were observed for Leishmania infection from one to ten days after the bloodmeal. We followed the bloodmeal digestion and compared the infection rate using using phase contrast microscopy, Giemsa-stained gut smears and transmission electron microscopy. Parasite density and distribution were observed in fresh dissected guts. Then, the tissue was homogenized and released parasites were counted in a haemocytometer. The infection rate and the colonization of the gut by the parasites were observed until 10 days after the infected bloodmeal. Transformation from amastigotes to promastigotes occurred rapidly and developed through as many parasite forms as: procyclic, nectomonad and haptomonad. Besides these forms, we also observed the common presence in the gut of degenerated forms of promastigotes, which usually appear after 4 days after the infection. We also observed parasites distributed all over the hindgut and Malpighian tubules in heavy infected flies. After five days, only small number of flies were able to keep the infection. The haptomonad and dividing short promastigotes are the predominant forms of promastigotes at the 6 day of infection. The development of the infection and the anterior migration of L. amazonensis in L. migonei appear to be similar to that described in natural host. Additional studies are being carrying out in order to define the ability of infection of the L. migonei for other Leishmania spp. and its epidemiology role in natural conditions.

098

INSULIN LIKE GROWTH FACTOR-1 INFLUENCES THE PATHOGENESIS OF LEISHMANIA (VIANNIA) PANAMENSIS INFECTION IN BALB/c MICE

Gomes, C.M.C ^1,3.**, Goto, H². ,Laurenti, M.D. ¹, Matta, V.L.R. ¹, Corbett, C.E.P.¹ & Gidlund, M.¹ Dep. Patologia¹ e Dep. Medicina Preventiva² Faculdade de Medicina da Universidade de São Paulo, Caixa Postal 292, S. Paulo, SP; ³Universidade Federal do Maranhão, S. Luis, MA, Brazil.

Many host-related non-specific factors are likely to play an important role in the Leishmania infection in the course of infection. We previously reported in vitro effect of IGF-1 on Leishmania promastigotes and amastigotes. In this study we evaluated the in vivo effect of IGF-1 on the course of infection with L.(V.) panamensis.

BALB/c mice were injected with 10⁵, 5x10⁵, 10⁶ and 10⁷ stationary phase L.(V.) panamensis promastigotes pre-incubated with or without IGF-1 (50ng/ml) in the foot pad and followed during 120 days. At 3, 24, 48 h, 7 days and one month post infection (PI) the specimen from the inoculation site was taken for histopathologic quantitative morphometric studies. The parasites pre-incubated with IGF-1 induced significantly bigger lesion from 9 weeks PI and onwards with all groups. The morphometric analysis of the skin of mice injected with parasites pre-incubated with IGF-1 showed a significantly higher number of neutrophils at 3 h PI and of mononuclear cells at 48 h, 7 and 30 days PI. In addition the phagocytosis of Leishmania was significantly higher both by polymorphonuclear neutrophils at 3 h PI and by mononuclear cells at 24, 48 h and 7 days PI.

IGF-1 plays an important role in the in vivo L.(V.) panamensis infection. The effect can be directly on parasites but in addition induces a more pronounced cellular infiltration and phagocytosis.

Supported by: LIM/HC-FMUSP, CNPq, FAPESP

099

HISTOPATHOLOGY OF THE KIDNEY IN DOGS NATURALLY INFECTED WITH LEISHMANIA (LEISHMANIA) CHAGASI FROM TEREZINA CITY IN PIAUÍ STATE, BRAZIL

^1,2Costa, F.A.L.**, ¹Klein, R.P. & ²Guerra, J.L.

¹Dep. de Clínica e Cirurgia Veterinária, Centro de Ciências Agrárias, UFPI, Terezina, 64049-550 - Brazil; ²Dep. Patologia, FMVZ-USP.

Canine visceral leishmaniasis (VL) is endemic in Terezina city in Piauí state of Brazil. Presently the dogs with positive serology for VL have been captured and eliminated by the Centro de Controle de Zoonoses do Estado. We studied the alterations in kidney in canine VL from this population since reports and systematic studies on this subject are scarce.

Thirty dogs with positive serology for VL were studied. 22 animals (73.3%) presented clinical signs of the chronic disease and 8 (26.6%) did not show any signs or symptoms. In order to confirm the dignosis cultures from bone marrow, spleen and popliteal lymph nodes were done as well as the characterization of the parasites. Fragments of kidney were taken and fixed in phosphate buffered 10% formaldehyde upon perfusion through carotid artery. Specimens were embedded in parafin and stained by Haematoxilin-Eosin (H&E), PAS and Masson dyes for histopathological analysis.

All animals showed glomerular and interstitial changes. The glomerular alterations were glomerulonephritis characterized as: minimal changes (N= 3, 10%), chronic (N=3, 10%), mesangioproliferative (N=10, 33,3%) and menbranoproliferative (N=14, 46,6%). The interstitial chages were characterized by mononuclear inflammatory infiltrate with different intensity: discreet in 18 animals (60%), moderate in 7 animals (23,3%) and intense in 5 animals (16,6%). The inflammatory reaction presented periglomerular and peritubular distribution. In 9 animals we observed the presence of hyalin casts and in 21 (70%) tubular degeneration.

We observed intense renal involvement in dogs naturally infected with L.(L.)chagasi same times even in those without any clinical signs of disease.

Supported by PICDT-UFPI.

100

ANTI-LEISHMANIAL DRUG (GLUCANTIME^®) HAS OXIDATIVE EFFECT ON LOW DENSITY LIPOPROTEIN (LDL) AND PROGRESSION OF VISCERAL LEISHMANIASIS.

Lindoso, JAL^1,2,3 Gidlund, M² & Goto, H.^2,4

1. Institute of Infectology Emílio Ribas 2. Dept. Pathology , 3. Dept. of Medical Clinic (Allergy and Imunopathology) & 4.Dept. Preventive Medicine of University of São Paulo Medical School, São Paulo, SP, Brasil.

Leishamaniasis are disease caused by several species of Leishmania. The drugs used in treatment are pentavalent antimonials. In Brazil the most used drug is meglumine antimoniate (Glucantime^®), but the mechanism of action is not well known. We have shown previously a correlation between the presence of oxidized products of LDL and the progression of infection (Lindoso et al-Mem Inst Oswaldo Cruz 91 Suppl. :175,1996). In addition we shown an oxidative effect of Glucantime^® on LDL mediated by cells (Lindoso et al-Mem Inst Oswaldo Cruz 91 Suppl. :195,1996). In this work we have studied the alterations of LDL in sera of hamsters treated with antimonials. Hamsters were infected intraperitoneally with 2 x 10⁷L. (L.) chagasi and treated with Glucantime^® (100 mg/g/day) during 15 days beginning at 45 days post infection. We measured the degree of lipid peroxidation using TBARS and we used the ELISA to detect oxidizied LDL. We observed that inicially the Glucantime^® decreased the parasite burden in liver and spleen but I was not completelly efficient in the treatment of visceral leishmaniasis in hamsters. In addition we demostreated that drug induces the increased oxidation of LDL in the group of hamsters treated at 60 days post infection (a day after the end of treatment). The presence of oxidized LDL changes the ration between parasite burden in liver and spleen and the the oxidation is high the ratio between parasite burden in liver and spleen become low. The data suggest that the drug influences the inflammatory process and induces oxidation of LDL with consequent effec on parasite burden.

Supported by: LIM/HC FMUSP, FAPESP an CNPq

EVALUATION OF DOGS AS SENTINELS OF THE TRANSMISSION OF TRYPANOSOMA CRUZI IN A RURAL AREA OF NORTHWESTERN ARGENTINA

CASTAÑERA MB,¹ LAURICELLA MA,² CHUIT R,³ & GÜRTLER RE.¹¹Departamento de Biología, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, 1428 Buenos Aires, Argentina. ² Instituto Nacional de Parasitología "Dr. Mario Fatala Chabén". ³ Dirección de Epidemiología de la Nación. moni@bg.fcen.uba.ar

Dogs are the main domestic reservoirs of T. cruzi in the argentine Chaco region. In the context of a vector control or elimination program, dogs may serve as sentinels of vector-mediated transmission of T. cruzi if infections acquired through other possible routes (vertical, ingestion of infected mammals, and horizontal) could be excluded. With this purpose in mind, we studied prospectively the dog population from Amamá and neighboring villages (Santiago del Estero, Argentina) before spraying of residual insecticides (in 1992) and during vigilance (1994 and 1996). House reinfestation was monitored through biosensor boxes, flushing-out searches and house-dwellers' captures. A house-to-house census of all dogs and interviews with the owners was carried out each 6 months to record demographic, epidemiologic and behavioral data on each individual dog. Serum samples were taken from nearly 70% of dogs in each year and tested by ELISA, indirect immunofluorescence antibody assay, and indirect hemagglutination test (Polychaco S.A., Argentina); seropositive refers to samples reactive by at least two different serologic tests in any one year. The overall prevalence of seropositivity for T. cruzi fell from 65% in 1992 to 39% (70/182) in 1994 and 15% (36/237) in 1996. No seroconversions of dogs older than 2 years of age were detected between 1994 and 1996. Among dogs less than 2 years of age (born after elimination of Triatoma infestans), the seroprevalence was 12% (10/87) in 1994 and 4% (6/148) in 1996. Eleven predictors of the ocurrence of a seropositive native dog born during vigilance were analyzed using multiple logistic regression with EGRET software. Significant predictors of the adjusted odds of being seropositive were the total number of Triatoma guasayana collected in bedroom areas (for 1994 data), the dog's mother status of infection and the number of seropositive dogs cohabiting with each dog (for 1996 data). The evidence suggest that new cases of infected dogs may arise from different routes of transmissions, and implicates T. guasayana as a secondary vector during vigilance.

This study was supported by the Rockefeller Foundations and the University of Buenos Aires.

102

EPIDEMIOLOGY OF AMERICAN CUTANEOUS LEISHMANIASIS (ACL) IN THE STATE OF SÃO PAULO. HYPOTHETICAL PATTERNS OF LEISHMANIA (VIANNIA) BRAZILIENSIS CIRCULATION DURING THE LAST 102 YEARS

Tolezano, JE, Araújo, MFL, & Taniguchi, HH

Seção de Parasitoses Sistêmicas - Instituto Adolfo Lutz - São Paulo-SP

It has exactly been 102 years ago, BREDA in Padova, Italy, had done clinical descriptions of "cutaneous affections", nowadays recognized as ACL, in Italian people who went back from Brazil, from inland of São Paulo State and from regions where the new agriculture and extrativism frontiers were opened. Since then, many informations have been produced on ACL, including discovery of endemic areas, clinical aspects, diagnosis, immunologic and epidemiological knowledge. In regard to the circulation of Leishmania (Viannia) braziliensis, the main agent of ACL in São Paulo, many obscure points remained unknown, and from them we can point out the primary and/or secondary vectors, the wild reservoir and/or vertebrate sources of infections to man. Examining the scientific literature, and considering the Knowledge produced from extensive epidemiologic studies, the authors reached to an hypothetical model in the attempt to explain the circulation of L.(V.) braziliensis in the last 102 years of ACL in the State of São Paulo. Basically we recognized two different patterns of risk of infection in distincts landscape, and assumed the participation of different sandflies vectors and reservoirs, and/or sources of infection. By the former pattern, registered until the middle of this century, the risk of aquisition of ACL were intimately related with penetration in natural forested environment or recently deforested, and by the contact with vectors of Leishmania (montained at enzootic level), especially Lutzomyia whitmani. By this pattern, is possible to deduce that the risk of infection were high, but restrict to a short time, until the endemic region were deforested and new commmmunities were established. By the second pattern, since 1940-1950 and during the second half of this century the risk of infection with L.(V.)braziliensis can be considered greater as those verified by the first pattern that had occupational connotation. Even though we consider a low disponibility of vertebrate sources of infection; this higher risk is consequence of a higher exposition of human communities, who living in areas with natural focus participate in the ecotope where L.(V.)braziliensis circulate, problaby transmitted by Lutzomyia intermedia holder of a great ecological valency.

103

EPIDEMIOLOGY OF AMERICAN CUTANEOUS LEISHMANIASIS (ACL) IN THE STATE OF SÃO PAULO. USE OF PARTICULATED ANTIGEN OF LEISHMANIA (VIANNIA) BRAZILIENSIS IN CANINE INQUIRIES IN DIFFERENT ENDEMIC AREAS

Tolezano, JE, Taniguchi, HH, Araújo, MFL, Bisugo, MC, Cunha, EA, Elias, CR and Larosa, R.

Seção de Parasitoses Sistêmicas do Instituto Adolfo Lutz- São Paulo-SP

Many researches have questioned about the involvement of domestic animals as sources of infection to man in endemic areas of ACL in the Southeast and South region of Brazil. FALQUETO et al., 1986, 1991 and 1993, in the State of Espírito Santo, had confirmed the role of dogs as "reservoirs" of L.(V.)braziliensis in the ancient zone covering atlantic forest, finding high rates of infection among dogs, around 17.0%. For the State of São Paulo, we have the same concern in relation to the vertebrate source of infection, probably represented by dogs. In this study we carried out two inquiries in order to investigate the Leishmania infection in canine population: In Itupeva (Jundiaí region) and Eldorado Paulista (Ribeira River Valley Region) cities which are all endemic areas. For the first time it was employed in prospectives studies the skin test (ST) with particulated antigen of L.(V.)braziliensis, and indirect immunofluorescence test (IIT) with antigens of L.(V.)braziliensis, L.(L.)chagasi and L.(L.) amazonensis was used in parallel. In Itupeva, from 56 inquired dogs, 10 (17.9%) were reactive for at least one of the two tests, when accepted any value of reactivity, and 4 out 56 (7.1%) when considered ST equal or higher than 5 mm or IIT equal or higher than 1:20. In Eldorado Paulista, only 3 out 77 (3.9%) dogs revealed some reactivity, all of them below 5 mm in SK and below 1:20 in IIT. These results indicate as promising enough and very practical for using the ST under field conditions for trial of natural infection of L.(V.) braziliensis for animals suspected at any importance as source of infection. By these studies even it could be minor the participation of dogs in maintenance of parasite in the nature in Eldorado Paulista, the data observed in Itupeva strengthen the necessity of better knowledge about involvement of dogs in the Leishmania circulation. Probably dogs play role in different degrees of relationship with Leishmania in different regions. We believe that such results would be fundamental for definition of control strategies.

Partially supported by PCDEN/FNS

104

EDENTATES AS A POSSIBLE RESERVOIR OF L. (V.) BRAZILIENSIS IN AN ENDEMIC AREA OF RIO DE JANEIRO

Pirmez C, Oliveira-Neto MP, Franco A, Meneses C, Rangel E, Mayrink A, Silva-Gonçalves, AJ, Fernandes O & Grimaldi G.

Hospital Evandro Chagas, Dept Immunology, Dept. Entomology, Dept Tropical Medicine and Dept Biochemistry & Molecular Biology, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brasil.

Despite the fact that L. (V.) braziliensis is the most important ethiological agent of mucocutaneous leishmaniasis in Brazil, there are many gaps in our knowledge concerning the natural mammalian hosts of the sylvan cycle of this particular species. Searching for wild mammal reservoirs of Leishmania in an endemic area of Rio de Janeiro (Mesquita), Brazil, we had the opportunity to examine seven sloths (Bradypus variegatus) and three opossum (Didelphis marsupialis). Whole blood was taken by venopuncture using vacutainer tubes containing EDTA. Genomic DNA was extracted from 500 µl of whole blood by column chromatography (Pharmacia Biotech - Uppsala, Sweden) following the manufacturer's instructions. The hot start PCR was performed with a pair of oligonucleotides that amplify the conserved region of the minicircle molecule of Leishmania. The 120 bp expected product was detected by agarose gel electrophoresis in three sloth samples. Hybridization with a PCR amplified conserved region of L. braziliensis MHOM/BR/75/2903 and L. amazonensis IFLA/BR/67/PH8 as molecular probes confirmed that the three samples belong to the Viannia subgenus.

The area is a typical representation of the epidemiological features that predominates nowadays in most brazilian endemic regions. The primary forest had been cleared for agricultural development, and thus drastically reducing the population of wild animals. These edendates were found in this endemic area near houses where human and dogs are infected by Leishmania, probably due to the recent discontinuity of palm-tree plantation. Two species of the Viannia subgenera, L. (V.) guyanensis and L. (V.) panamensis have edentates (sloths and/or anteaters) as their primary vertebrate hosts. Our results suggest that edentates can also be a primary reservoir for L. (V.) braziliensis parasites in the examined endemic area of Rio de Janeiro.

Supported by FAPERJ

105

CONTRIBUTION TO THE STUDY OF BENTHIC CILIATES (HYPOTRICHIDA, PROTOZOA) FROM GUANABARA BAY, RIO DE JANEIRO, BRAZIL.

Dias, C.T.M. & Silva Neto, I. D. da

Laboratório de Protistologia, Dept^o de Zoologia, IB/CCS, Universidade Federal do Rio de Janeiro, Avenida Brigadeiro Trompowski, s/n^o, Ilha do Fundão, Rio de Janeiro, RJ, Brasil.

This study is being realized at Guanabara Bay, located in the metropolitan area of Rio de Janeiro state, Brazil, which receive pollutants arising from discharge of organic matter and industry liquid waste. This area has an important ecosystem with multiples uses like navigation, water supply and recreation, and several academic studies are being doing, with all compartment of this ecosystem. We are observed the ciliates from benthic sand beaches, where this organisms play an important role like bacteria predators. We collected samples from superficial sediments from two stations, Urca e Ilha do Fundão, little sub-samples were withdrew and observed in humid chamber at stereoscopic microscopic. We observed an large variation in the ciliate faunula. The ciliates were took and the species firstly studied was those more abundant and easily maintained in culture in Petri dishes at the laboratory. The predominant species in these conditions belong to order Hypotrichida, family Euplotidae. Organisms from these cultures were separated and analysed using the silver impregnation technique, modified and improved by several authors: Protargol, which required the silver proteinate as the impregnant agent and wet silver method, were used silver nitrate as the impregnant agent. The permanent slides were prepared for detailed studies using microphotographs and schematic drawings at the microscopic equipped with bright-field, showing the infraciliature structures from the somatic and oral cortex, very important from an specific identification. Drawings with organisms in vivo were doing too. These are the first results from ciliates hypotrichous that occur in the benthic ecosystem from Guanabara Bay. The classification will be discussed and detailed descriptions of some species, photomicrographs and drawings will be presented.

Supported by CAPES, CNPq, FAPERJ and CEPG-UFRJ.

106

THE PRIMITIVE TRANSMISSION CYCLE OF AMERICAN CUTANEOUS LEISHMANIASIS (ACL) AND THE LINK WITH THE EPIDEMIOLOGICAL CHAIN IN PERI-DOMESTIC HABITATS IN RIO DE JANEIRO, BRAZIL

Abstract not received.

107

THE TRANSMISSION CYCLE AND THE HETEROGENEITY OF TRYPANOSOMA CRUZI ISOLATES DERIVED FROM MARSUPIALS (DIDELPHIS MARSUPIALIS AND PHILANDER OPOSSUM) CAPTURED IN TERESÓPOLIS, RIO DE JANEIRO.

Pinho, A.P.; Cabrera,B.A.; Gomes-Cardoso;L.; Cupolillo,E. & Jansen, A.M.

Departamento de Protozoologia, Lab. de Biologia de Tripanosomatídeos. Intituto Oswaldo Cruz. FIOCRUZ. Av.Brasil 4365, Manguinhos, cep.21045-900, Rio de Janeiro, RJ., Brasil.

Twenty seven T. cruzi isolates from naturally infected marsupials ( Didelphis marsupialis and Philander opossum ) and triatomid bugs (Rhodnius prolixus ) captured in Teresópolis , Rio de Janeiro were characterized by biological and biochemical parameters. The course of the experimental infection in swiss mice was followed up and the electrophoretic profile of the following enzimes analysed: GPI (E.C.5.3.1.9), G6PDH (E.C.1.1.1.49), ME ( E.C.1.1.1.40), IDH (E.C.1.1.1.103) and MDH (1.1.1.37). Metacyclic forms from each isolate obtained in axenic medium (10⁵) were inculated intraperitoneally in male swiss outbred mice weighting 18g. The parasitemia was followed up every other day by fresh blood smears examination and/or countings in a Neubauer chamber. The course of the experimental infection of the mice allowed us to discriminate the isolates in three groups: a - Isolates resulting in high patent parasitemia and no mortality ( eight D. marsupialis and two P. opossum isolates); b - Isolates resulting in subpatent parasitemia and no mortality ( the seven bug isolates and two D. marsupialis isolates); c - Isolates resulting in high partasitemia and high mortality rate: eigth P. opossum and two D. masupialis isolates). Moreover, the 27 isolates could be arranged in 9 distinct zymodemes patterns. The R. prolixus isolates shared only two zymodemes with the isolates from D.marsupialis and none with the P. opossum isolates, strongly suggesting that in in that given area, R. prolixus is not involved in the transmission of T.cruzi among P. opossum and is not an important vector for D. marsupialis. Only two zymodemes were shared by the marsupial isolates. No correlation could be established among the biological and biochemical parameters tested, such as: prepatent and patent period, parasitemic peak and mortality and zymodemes data. Our data show that the collected R.prolixus were involved mainly with the D. marsupialis transmission cycle and that the D. marsupialis isolates displayed a higher diversity than the P. opossum isolates. It is worthwhile to mention that in experimental conditions D. marsupialis is a stricter biological filter than P.opossum

Our results strengthen our previous propositions that the transmision cycle of T. cruzi in the sylvan environment, can be rather independent even among closely related mammals.

PAPES/FIOCRUZ, CNPq

108

THE KYNETIC OF TRANSMISSION OF TRYPANOSOMA CRUZI AMONG DIFERENT GROUPS OF LEONTOPITHECUS ROSALIA (PRIMATA, CALLITHRICHIDAE) IN THE ATLANTIC COASTAL RAINFORESTS OF BRAZIL.

Lisboa, C.V. ⁽¹⁾; Martins, A. ⁽²⁾; Breen, J.O. ⁽¹⁾; Mangia, R.H. ^{(3 )} & Jansen, A.M. ⁽¹⁾.

(1) Laboratório de Biologia de Tripanosomatídeos, Instituto Oswaldo Cruz, Caixa Postal 926, 21045-900, RJ, Brasil. (2) Associação do Mico-Leão-Dourado - Projeto de Reintrodução, Caixa Postal 109995, 28860-000, RJ, Brasil. (3) Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, Caixa Postal 926, 21045-900, RJ, Brasil.

The wild golden lion tamarin (Leontopithecus rosalia) is endemic to the Atlantic Coastal Rainforest of Rio de Janeiro, Brazil. An international program to conserve golden lion tamarins was initiated in 1983 in the Poço das Antas Biological Reserve (Rebio), Rio de Janeiro State. One of the objectives of this program is to reduce the probability of extinction of this species in the wild, through reintroduction of captive-born tamarins and translocation of wild-born tamarins. An aspect of this program includes the release of tamarins on privately-owned biological reserves (RPPN) surrounding the Rebio. Since 1995 we have examined 108 tamarins through Giemsa-stained blood smears, hemocultures and indirect immunofluorescence assay (IFAT) in the Rebio. Positive IFAT was observed in 49% of these, and the parasite was isolated from 35 individuals. The biological and biochemical characterization of these isolates were different from those derived from the other mammals in the reserve (Lisboa, 1996. Mem. Inst. Oswaldo Cruz, RJ, vol. 91, suppl.,1996). Because we consider the golden lion tamarin to be the principle natural reservoir of T. cruzi in the Rebio, we are studying the dynamic of the transmission cycle of tamarins reintroduced into the RPPN. Using the same methods described above in 65 tamarins captured on 4 RPPN's surrounding the Rebio, twelve individuals (18,46%) were found to be serologically positive by IFAT. Hemocultures to isolate the parasite are still in progress. The distribution of infected animals by RPPN, from farthest to closest to the Rebio, is the following: Rio Vermelho, 0% of 17 individuals; Santa Helena, 62,5% of 16 individuals; Dois Irmaos, 4,5% of 22 individuals and Igarapé, 9% of 11 individuos. Of these RPPN's, only Igarapé and Dois Irmaos share boundaries with the Rebio. Our results, although preliminary, suggest that independent cycles of T. cruzi may exist even within populations of the same species in close geographic proximity, which reinforces the idea of the existence of independent sylvatic transmission cycles. Additionally, the environmental conditions of certain RPPN's, and their effects on tamarin behaviors, may favor the transmission of T. cruzi within the tamarin population. It is worthwhile to consider that environmental conservation programs of the tamarin, as well as of other protected animals, may contribute to the dispersion of parasites such as T. cruzi within determined areas.

FIOCRUZ/PAPES/FAPERJ/IBAMA/AMLD

109

AMERICAN TEGUMENTARY LEISHMANIASIS (ATL) IN RIO DE JANEIRO STATE, BRAZIL: SEARCHING THE PUTATIVE L.(VIANNIA) BRAZILIENSIS VERTEBRATE HOSTS

¹Franco AMR, ²Rangel E, ¹Moreira CFS, ¹Mayrink AN,¹ Da Silva, A, ³Oliveira Neto MP & ¹Grimaldi Jr.,G.

¹Dept Immunol., ²Dept Entomol. & ³ Hospital Evandro Chagas - FIOCRUZ, CP. 926, RJ, Brasil

Wild mammals serve as reservoirs for most of the New World Leishmania, but there is increasing evidence that some of the human pathogenic Leishmania can be maintained in both sylvan and urban cycles (Grimaldi et al., 1989). In the case of L. (V.) braziliensis, the principal vertebrate hosts in the sylvan cycle have not been identified, but there is evidence that dogs, horses, and donkeys may serve as reservoir hosts in an urban cycle of this parasite (for review see Grimaldi & Tesh, 1993). During the first outbreak of ATL (from July 1984 to September 1986) in a periurban area (the municipality of Nova Iguaçu) of Rio de Janeiro city, Brazil, we examined 105 confirmed human cases of the disease. Domestic animals were easily found infected: 32% of the examined dogs and 30.8% of the examined equines were positive to the presence of Leishmania in cutaneous ulcerated lesions. Parasites isolates from human, dog and equines were identified as L. (V.) braziliensis (Oliveira Neto et al., 1988). The region corresponds geographically to the primitive area of the Atlantic forest in Brazil, located on the southeast coast of the country. Here we extend these studies, searching for wild mammal reservoirs of Leishmania sp. in the area under study. The survey of wild animals was carried out by means of parasitological diagnosis. As a first approach, we attempted to isolate flagellates from the putative Leishmania vertebrate hosts of the region, and then identify the species of the isolates. Of 10 wild animals, belonging to two mammalian orders [Bradypus variegatus (7) and Didelphis marsupialis (3)], we have failed to demonstrate leishmanial parasites in the tissues examined (peripheral blood and/or skin, liver and spleen) by using either the stained smear technique or other recommended procedures such as cultivation in vitro and/or inoculation of laboratory animals with the biopsed material. In conclusion, no evidence of a natural enzootic focus of the L. (V.) braziliensis infection, by parasitological methods, despite our continuos search for wild reservoirs, was determined in this study. However, serological techniques or the success of new molecular biology tools (e.g., hybridization identification or PCR technique, which requires recombinant DNA selection of specific sequences/probes) has been under investigation by our group to evaluate their efficacy to more practical issues such as the specific diagnosis of the disease or the molecular epidemiology of each Leishmania species.

(Supported by CNPq, FAPERJ).

110

EFFECTS OF TRYPANOSOMA EVANSI ON THE BLOOD CHEMISTRY AND HEMATOLOGY OF COATIS (NASUA NASUA) NATURALLY INFECTED IN THE PANTANAL, BRAZIL

Silva, R.A.M.S.;¹ Victório, A.M.;¹ Ramirez, L.; ¹ Dávila, A.M.R.,¹ Trajano, V.;² & Jansen , A.M.^2,

¹ Laboratório de Ecopatologia, EMBRAPA/CPA-PANTANAL, Rua 21 de Setembro, 1880, CEP: 79320-900, Corumbá, M.S., Brasil, E-mail: ramss@cpap.embrapa.br, ² Laboratório de Biologia de Tripanosomatídeos, Departamento de Protozoologia, Instituto Oswaldo Cruz, Rio de Janeiro, Brasil.

Equine trypanosomosis caused by Trypanosoma evansi is known in the Pantanal and subtropical areas of Argentina as "Mal de Caderas". T. evansi has the widest distribution of all species of trypanosomes and the greatest range of mammalian hosts. During the study of a wild reservoir of Trypanosoma evansi in the Pantanal, Brazil, forty coatis (Nasua nasua) were caught in three ranches located in the Nhecolândia subregion of Pantanal. Hematologic and blood chemistry values observed in infected coatis (30.0 %) are given in table. These data are indicative of the importance of coatis as a wild reservoir and the importance of this parasite as causing disease in coatis. The nature of the blood and chemistry alterations need to be elucidated and mechanisms proposed. This is the first report of T. evansi causing disease in coatis.

Hematologic and blood chemistry values of coatis (Nasua nasua) naturally infected by Trypanosoma evansi in the Pantanal, Brazil and non-infected coatis.

RBC count
x 10⁶/ mm³ WBC count
x 10³/mm³ Ht (%) Hb mg/dl Glucose mg/dl Albumin g/dl Infected
Captivity+
Semi-cativity+ 4.21 ± 1.13a
7.5 ± 2.00 b
5.5 ± 0.90 ^b 12.68 ± 4.68a
13.40 ± 4.50 ^a
8.10 ± 1.80 ^a 28.19 ± 5.27a
40.90 ± 6.20^b
30.80 ± 2.90 ^a 9.51 ± 1.290a
12.20 ± 3.30 ^a
9.70 ± 0.90 ^a 73.60 ± 44.53a
176.20 ± 38.40^b
176.20 ± 38.40 ^bc 1.18 ± 0.80a
4.50 ± 1.20 ^{b c}
4.50 ± 1.20 ^{b c} RBC: red blood cell; WBC: white blood cell; Ht:hematocrit; Hb: hemoglobin; +according to A.L.G. Pimentel (1994).
^{a, b, c} Values across a row with different letters are significantly different (P< 0.0005).

A QUESTIONNAIRE SURVEY TO DETERMINE AWARENESS OF VISCERAL LEISHMANIASIS AND RISK FACTORS FOR ITS TRANSMISSION AMONG RESIDENTS OF AN ENDEMIC FOCUS OF LEISHMANIA CHAGASI IN MONTES CLAROS, MINAS GERAIS

Alexander, B, Calaça¹, PF, Rios, E¹ & Vieira, E²

Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Caixa Postal 486, Belo Horizonte 31270-901 MG, Brazil; ¹Faculdade de Medicina, Universidade Estadual de Montes Claros, Avenida Dr. Rui Braga, Montes Claros 39401-089 MG, Brazil; ²CETEC, Fundação Nacional de Saúde, Montes Claros, MG, Brazil.

A questionnaire survey was done of 341 residents of eight neighbourhoods endemic for Leishmania chagasi in Montes Claros M.G., to determine their knowledge of visceral leishmanisis (VL) and its transmission by phlebotomine sand flies, as well as to identify risk factors for contact with Lutzomyia longipalpis. Previous studies (O. Genaro, unpublished data) using immunofluorescence antibody testing showed the prevalence of canine infection in these neighbourhoods to be between 3.6% (Independencia) and 14.2% (Jardim Panorama). Although 40.0% of the people interviewed knew how Le. chagasi was transmitted, only one was able to describe the insects that caused a biting nuisance in his house as sand flies rather than mosquitoes. None of the common names for sand flies used in Brazil (e.g. "cangalinha", "asa branca" or "mosquito de palha") that describe the appearance of the insect were known to the people interviewed. Forty people (11.8%) knew someone who had contracted VL and 93 (27.4%) had owned or knew of a dog that had died from the disease or been destroyed by the health authorities after VL diagnosis. Dogs were kept by between 25.0% (Vila Atlantida) and 60.0% (Alcides Rabelo) of households. Between 0 (Jardim Panorama) and 24.0% (Vila Mauriceia) of residents had chickens, while only 1.0% of people kept pigs in their yards. Ownership of pigs or chickens may be an important risk factor in acquiring VL, since animal shelters are attractive to Lu. longipalpis. This risk should be exacerbated for dog owners and this situation was seen in 23 households, with Vila Atlantida (16.7%) having the highest percentage of houses of this type. Collections made in one pigsty and one chicken house in Vila Mauriceia at the time of the survey were found to consist of 45.5% Lu. longipalpis, the remainder being Lu. intermedia (n = 57). Regular sand fly collections will be made in these and four other neighbourhoods of Montes Claros and correlated with the results obtained from our questionnaire survey to define risk factors associated with presence of Lu. longipalpis, as a first step towards implementing a control programme based on community participation.

Apoiado pela Fundação Nacional de Saúde.

112

ABORTIONS CAUSED BY TRYPANOSOMA VIVAX IN BOVINES FROM THE PANTANAL OF POCONÉ, MT, BRAZIL.

Silva, R.A.M.S¹ ; Dávila, A.M.R.¹ ; Ramirez, L.¹ & Pellegrin, A.O.²

¹Laboratório de Ecopatologia, EMBRAPA/ Centro de Pesquisa Agropecuária do Pantanal (CPAP), Rua 21 de Setembro, 1880, 79320-900, Corumbá, MS, Brazil. ² Lab. de Bacteriologia, EMBRAPA/CPAP. Author for correspondence. ramss@cpap. embrapa.br.

According to Ogwu & Njoku (1987 Veterinary Parasitology 24:25-33) Trypanosoma vivax infected non-pregnant heifers and heifers in the third trimester of pregnancy developed a more severe form of the disease than pregnant heifers in the first and second trimesters of pregnancy. Calves born of infected heifers had enlarged spleens and lymph nodes and trypanosomes are present in their blood, providing evidence of transplacental transmission (Ogwu et al, 1986 Theriogenology 25:383-398).

In the begining of 1995, several outbreaks of fever, anemia, progressive weakness, loss of condition, loss of appetite, lethargy, substantial weight loss within a relative short period of time, anemia and progressive emaciation, abortion and death of bovines occured in the Pantanal of Poconé. Ten of the twenty-nine (34.48%) bovines (Bos taurus taurus x Bos taurus indicus) from nine cattle ranches were infected by T. vivax. All ranches presented positive animals (Silva, et al. 1996 Mem Inst Oswaldo Cruz 91:561-562). Seven of 9 ranches were investigated for records of abortions. The ranchs were designated as R1 to R7, respectively. The total number of bovines by ranch was 200, 400, 400, 250, 450, 400 and 300 in R1-R7, respectively. The percent of abortions was 8, 0, 1.5, 1.6, 1.3, 0.5 and 0.6 in R1-R7, respectively. All abortions occured in the third trimester of pregnancy. Our results are similar with those recorded by Ogwu & Njoku (loc. cit.) in Nigeria because severe clinical signs (e.g. blindness) were observed in cows from the Pantanal in the third trimester of pregnancy.

113

BOVINE TRYPANOSOMIASIS DUE TO TRYPANOSOMA VIVAX IN THE GERMAN BUSH PROVINCE, BOLIVIA

Silva, R.A.M.S. ¹, Dávila, A.M.R. ^1,2, Pereira, M.E.B. ², Ramirez, L.^1,2, Sanches, W.³ & Gutiérrez, J.L.⁴

¹ Laboratório de Ecopatologia, EMBRAPA, Centro de Pesquisa Agropecuária do Pantanal, Rua 21 de Setembro, 1880, CEP 79320-900, Corumbá, MS, Brasil. E-mail: ramss@cpap.embrapa.br

² UFMS/Centro Universitário de Corumbá, Corumbá, MS, Brasil³ Federación de Ganaderos de Santa Cruz, Puerto Suarez, Bolivia. ⁴ Clinica Veterinária Pantanal, Av. Santa Cruz, 32, Puerto Suarez, Bolivia.

Trypanosoma vivax is found throughout the tsetse belt in Africa. It has spread to other parts of Africa, Central America, South America, the West Indies and Mauritius. In 1919 T. vivax was reported in the New World for the first time in French Guyana and later in other parts of South America, Central America, and some Caribbean islands. Bolivia is a sub-tropical country located in the center of South America. It has nine departments. The Santa Cruz department is considered one of the most important livestock regions, maintaining a population of 1,598,957 bovines. The German Bush province is located in the extreme south of Santa Cruz department in the border of Paraguay.

In November 1996, we received preliminary reports of anemia and abortions in cattle from bolivian veterinarians of the German Bush province. In February 1997, 87 bovines belonging to 4 ranches of German Bush, were bled from their jugular vein using a vacuum system (Vacuum II, Labnew, Campinas, Brazil). The sampled animals, all nelore purebred and crossbreeds (Bos taurus taurus x Bos taurus indicus) between 1 and 9 years old (mean 7 years). The diagnosis was made using the microhematocrit centrifuge test (MHCT). Blood from each sample and the concentrated parasites in the buffy coat of microhematocrit tubes were also used to prepare thin smears. The trypanosomes were identified based on morphological and biometrical data. The clinical signs observed were fever, anemia, abortion, progressive weakness, substantial weight loss in relative short time, and progressive emaciation and lymphonode enlargement. Some animals registered a hematocrit as low as 17%. Thirty-nine of the eighty-seven bovines examined by microhematocrit test were infected by T. vivax (47,82%). The results of this study suggets that T. vivax represent a serious impact to the economy of the region.

114

OUTBREAKS OF BOVINE TRYPANOSOMIASIS BY TRYPANOSOMA VIVAX IN THE MATO GROSSO DO SUL STATE, BRAZIL

Silva, R.A.M.S. ¹; Ramirez, L. ¹; Dávila, A.M.R.¹; Ferreira, M.J. da S.² & Sahib, C.A. ²

¹ Laboratório de Ecopatologia, EMBRAPA/Centro de Pesquisa Agropecuária do Pantanal (CPAP), Rua 21 de Setembro, 1880, 79320-900, Corumbá, MS, Brazil. Author for correspondence. ramss@cpap. embrapa.br. ²Clínica Veterinária Corumbá, MS, Brazil.

Trypanosoma vivax is found throughout the tsetse belt in Africa. It has now spread to others parts of Africa and Central America, South America, the West Indies and Mauritius. In 1919, T. vivax was reported in the Americas for the first time in French Guyana and later in others parts of South America, Central America, and some Caribbean islands. In 1972, T. vivax was reported in a water buffalo (Bubalis bubalis) in the vicinity of the city of Belém, Pará State. In 1996, T. vivax was reported in Poconé, Mato Grosso, Brazil. We report here, for the first time, bovine trypanosomosis due to T. vivax in Mato Grosso do Sul. In the begining of November 1996, several cases of fever, anemia, progressive weakness, loss of condition, loss of appetite, lethargy, substantial weight loss within a relativly short period of time, progressive emaciation and death of 20 bovines in Paiaguás and 5 in the Nabileque subregions of the Pantanal located within the state of Mato Grosso do Sul were reported. Some animals presented a hematocrit as low as 18%. The mean value of hematocrit observed was 22.66 %. T. vivax was diagnosed by microhematocrit test and thin blood smears in 80.95% (17/21) and 50% (1/2) bovines from Nabileque and Paiaguás subregions respectively. T. vivax potentially represents an important bovine disease in Mato Grosso do Sul. However, more studies will be necessary to determine the epizootiology of T. vivax and the impact of the disease on the economy of the region.

115

OCCURRENCE OF CANINE TRYPANOSOMIASIS DUE TO TRYPANOSOMA EVANSI IN THE PANTANAL, BRAZIL: HEMATOLOGIC DISORDERS

Ramirez, L. ^1,2.; Souza, S.S. ^1,2, Brandão, M.C. ^1,2, Silva, R.A.M.S.² & Dávila, A.M.R. ^1,2

¹Universidade Federal de Mato Grosso do Sul, CEUC/DAM, Av. Rio Branco 1270, Corumbá, MS, Brasil ²Laboratório de Ecopatologia, EMBRAPA, Centro de Pesquisa Agropecuária do Pantanal, rua 21 de Setembro, 1880, CEP 79320-900, Corumbá, MS, Brasil.

According to Losos (Infectious Tropical Diseases of Domestic Animals. Essex: Longman Scientific & Technical, p.938, 1986) hematological alterations are the most consistent findings in salivarian trypansomiasis. The pathogenesis of T. evansi and other trypanosomes of the subgenus trypanozoon is characterized by rapid loss of weight and varying degrees of anemia. Leukopenia has been reported in trypanosomosis and is attributed to reduced myelopoiesis (Jenkins, G. C & Facer, C. A. Hematology of African Trypanosomiasis, in Tizard, I. Immunology and Pathogenesis of Trypanosomiasis. Boca Raton, Florida p. 13-44, 1985). In July 1997, an outbreak of canine trypanosomiasis occured in a ranch located in Pantanal of Nhecolândia subregion. Blood samples were taken from 6 sick dogs. The dogs were bled from their radial vein using a vacuum system (Vacuum II, Labnew, Campinas, Brazil). The hematocrit (Ht) was measured using the standard method, and the red blood cell count (RBC) and total white blood cell count (WBC) were obtained using Neubauer chamber. The diagnosis of trypanosomiasis was made using the microhematocrit centrifuge test. The concentrated parasites in the buffy coat of micro hematocrit tubes were also used to prepare thin smears. Three blood samples (50%) from 6 dogs were positive. The parasites were identified based on morphological and biometrical data. The mean hematologic values observed in infected dogs were: RBC: 1.11 x 10⁶ mm³; WBC: 1.9 x 10³ mm³; Ht: 20%. The dogs presented anemia characterized by a low erythrocite count and decreased hematocrit as well as leukopenia. Several erythrocyte abnormalities were observed in the blood of infected dogs: microspherocytes, acanthocytes, stomatocytes, drepanocytes, dacrocytes and keratocytes. Anisocytosis and poikilocytosis were present. Adhesion of erythrocytes to trypanosomes in circulanting blood was also observed. We believe that several erythrocyte abnormalities contributed of to the increased red cell destruction.

116

THE CILIATES FROM PIRATININGA LAGOON, NITERÓI-RJ.

Joppert, F.^* * Mestrado Zoologia, Museu Nacional-UFRJ & Silva Neto, I.D. da

Lab. de Protistologia, Dept. de Zoologia, IB, UFRJ, Rio de Janeiro, 21941-590 RJ, Brasil.

The Protist kingdom is a very large and an extremely diversified one. The Phylum Ciliophora have more than 8000 described species and tend to have a cosmopolitan distribution. The salt and brackish lagoon located at the Brazilian littoral can show a great number of ciliates in which their main role is to transfer the energy from the bacteria to the higher levels of the food chains. The Piratininga lagoon receives in its water domestic waste discharges that can cause several problems to the ecosystem, like eutrophication. It is located in a very important area which presents high land speculation reflecting in a water body reduction due to landfills.

The samples of Piratininga Lagoon were collected several times with flasks and taken to the laboratory, where they were put into Petri dishes with mineral water and rice. After one week the ciliates were examined in stereoscopic microscopic and separated with micropipettes and fixed in Stieve`s fluid according to Bodian Silver Impregnation Technique, 1937, modified by Tuffrau, 1967, improved by Foissner, 1991 (Protargol technique) and Silva Neto, 1996.

The results showed the structural aspects like oral apparatus, somatic ciliature including the cortical structures such as basal bodies and fibril systems, and the nuclear apparatus as macro and micronuclei. We determined four ciliate species: Euplotes woodruffi, Urostyla sp., Steinia sp. and Paramecium sp.

Supported by CAPES, CNPq, FAPERJ and CEPG.

117

LEISHMANIA SPECIES IDENTIFICATION FROM AUTHOCHTONOUS CASES OF HUMAN CUTANEOUS LEISHMANIASIS IN SANTA CATARINA STATE, SOUTHERN BRAZIL.

Steindel, M.*; Shaw, J.J.**; Ishikawa, I. A.Y.**; Carvalho Pinto, C.J.*; Toma, H.K.*; Grisard, E.C.* & Lima, J.H.*

*Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Catarina. Caixa Postal 476, 88040-900, Florianópolis, SC, Brasil, e-mail: mip1mst@ccb.ufsc.br; **Departamento de Parasitologia ICB, USP, Av. Prof. Lineu Prestes 1374, 05508-900 São Paulo, SP, Brasil.

An outbreak of human cutaneous leishmaniasis was described in two municipalities (Quilombo and Coronel Freitas) in the west region of Santa Catarina State (São Thiago & Guida, Rev. Soc. Bras. Med. Trop. 23(4): 201-203, 1990). Eleven authoctonous cases were confirmed by epidemiology, Montenegro skin test, Giemsa stained smears and response to specific treatment with Glucantime^Ò. Two strains (MHOM/BR/89/JSC89-H1 and MHOM/BR/89/JSC89-H2) were isolated from lesions of two adult patients by inoculation in hamsters and subsequent isolation in LIT medium. The parasites were maintained at 27ºC by weekly passages in NNN+LIT. Recently, another authoctonous case was detected at Chapecó municipality on the same region. This strain (MHOM/BR/96/LSC/96-H3) was isolated by the same way and maintained in Schneider's medium at 27ºC after isolation. Parasites maintained in NNN+LIT or Schneider's medium were harvested in the exponential growth phase and washed 3 times in phosphate buffered saline (PBS) pH 7.2 at 1,500 g. Flagellates were ressuspendend in PBS to a final concentration of 10⁶ parasites/ml and 10ml of this suspension was applied to each immunofluorescence slide wells. After air dring and fixing in cold acetone, the samples were incubated with specific monoclonal antibodies (Mbas). Thereafter, an indirect immunofluorescent avidin/biotin or fluorescein labelled antibody system was used for Leishmania identification. Two different species of Leishmania were recognized by Mbas. MHOM/BR/89/JSC89-H1 and MHOM/BR/89/JSC89-H2 strains belong to the Leishmania amazonensis complex and MHOM/BR/96/LSC/96-H3 strain was recognized by B-16 L. braziliensis Mab. These results show that cutaneous leishmaniasis outbreak in the State of Santa Catarina are due to distintc Leishmania species and point out for the cutaneous leishmaniasis spreading in Brazil.

Supported by FNS and CNPq.

118

TOXOPLASMOSIS IN PREGNANT WOMEN FROM INSTITUTO FERNANDES FIGUEIRA - FIOCRUZ -RJ,IN A PERIOD BETWEEN OCTOBER , 1994 AND NOVEMBER , 1996 - EPIDEMIOLOGICAL ASPECTS.

FERREIRA, H.G.¹; ALEXANDRE, G. M. C.¹; COSTA, T. da ¹; VICENTE, R. T.¹; NOGUEIRA, M. C. A .¹; CUNHA, C. L. M. da ²; COSTA, M. C. F. L.²; UCHÔA, C. M. A.³; FERREIRA, M. do C.⁴ & AMENDOEIRA, M. R. R. A.¹.

1-Protozoologia, Instituto Oswaldo Cruz - FIOCRUZ - Av. Brasil, 4365, CEP: 21045-900, Rio de Janeiro - RJ, Brasil; 2- Instituto Fernandes Figueira - FIOCRUZ ; 3- Universidade Federal Fluminense; 4- Universidade do Rio de Janeiro.

The Toxoplasma gondii is a parasite with worldwide distribution able to infect a large variety of animal species including men. This fact can be attributed of diverse transmission mechanisms, some well known , such as: ingestion of cysts from undercooked or raw meats, or of oocysts from cat feces contaminating raw vegetables, earth manipulation, among others. This parasite is very important for pregnant women, as primary infection is often transmitted to the fetus , severally affecting them. The timing in the pregnancy period in which the woman had contact with the parasite is very important for the pathogenicity of infection. This investigation is proposed based on these facts; analysis of the epidemiological data of the parasites correlated with serological results from the pregnant women group. The seroepidemiological investigation has been done with 643 patients assisted at the Instituto Fernandes Figueira ambulatory in the period between october,1994 and november,1996. Of these, 467 pregnant women (72,62%) are from various regions within the city of Rio de Janeiro and 176 (27,37%) from regions periferic to Rio de Janeiro. Among the patients evaluated, 464 (72,12%) were seropositive. In the city of Rio de Janeiro a seropositivity of 73,44 % was registered, while in peripheral districts it was 68,75% (no significant difference) . When the risks of parasite transmission were considered , we registered the relative risk factors of the infection, as following: 1,65 risk for eating raw or undercooked meat (78,54%); 1,3 for patients who ate raw vegetables; 1,4 among pregnant women who had contact with cats; 1,6 those who handled earth (78,20%) and 1,5 in women whose occupation was domestic tasks (75,80%).

119

SEROPREVALENCE OF TRYPANOSOMA EVANSI IN EQUINES FROM THE NHECOLÂNDIA SUB-REGION OF THE PANTANAL

Dávila, A.M.R. ^1,2,3 & Silva, R.A.M.S. ¹

¹Lab. de Ecopatologia, EMBRAPA/CPA-Pantanal, Rua 21 de Setembro 1880, Corumbá, MS, Brasil, Caixa Postal 109. ²UFMS/CEUC/DAM, Corumbá, MS, Brasil. ³CNPq/RHAE studentship. Author for correspondence: alrida@cpap.embrapa.br

Equine trypanosomosis due to Trypanosoma evansi is one of the most important protozoal diseases found in the Pantanal region of Brazil. This parasite infects a variety of mammals. In the Pantanal, it has been found in horses, coatis (Nasua nasua), dogs, small wild rodents (Oryzomys sp.) and capybaras (Hydrochaeris hydrochaeris). Diagnostic tools for a sensitive and early diagnostic are necessary to study and understand the epizootiology of this protozoal disease in the Pantanal.

A survey to assess the prevalence of T. evansi in equines was carried out at the beginning of July on a ranch located in the Nhecolândia sub-region of the Pantanal. Three hundred and seventy-two animals were bled using a vacuum system. In this preliminary study, a total of 244 sera samples were tested using the Indirect Immunofluorescence Antibody Test (IFAT) for the diagnosis. The prevalence was 12,70% (31/244). The first 102 animals were examined for T. evansi using the microhematocrit centrifuge test (MHCT). The prevalence was 3.92 % (4/102).

Relative low prevalences may be obtained using MCHT for diagnosing trypanosomes in comparison to more accurate diagnostic tests. According to Monzón et al. (1990 Veterinary Parasitology 36:141-146) MHCT detected 71.1 % of natural T. evansi infections in horses from the Formosa province, Argentina. Our prevalences are similar with those described in the literature. Franke et al. (1994 Acta Tropica 58:159-169) found a T. evansi prevalence of 4.1 % in 364 horses from the Pantanal of Poconé region using standard parasitological methods. On the other hand, using the IFAT, Monzón et al. (1988 Arq Bras Med Vet Zoot 40:279-285) reported a prevalence of 19.3 % of T. evansi in horses from the Formosa department, Argentina, from 1983 to 1987.

Although we are showing preliminary results, the IFAT appears be more sensitivite than the MHCT. More sera samples are being tested and statistical analysis are being done in order to obtain prevalences of T. evansi according to sex, age and race.

120

EPIDEMIOLOGY OF LEISHMANIASIS IN THE BRAZILIAN AMAZONIAN BASIN

Maricleide de Farias Naiff; Ilner Souza e Souza¹; Roberto Daibis Naiff; Tobby Barrett; Luiz Eduardo de Carvalho-Paes³; Hooman Momen²; Elisa Cupolillo³ & Gabriel Grimaldi Jr.³

Instituto Nacional de Pesquisa da Amazônia; ¹Instituto de Dermatologia e Venerologia Alfredo da Matta, Manaus; ²Dept. de Bioquímica e Biologia Molecular and ³Dept. de Imunologia, Instituto Oswaldo Cruz - RJ

Both American cutaneous and mucocutaneous leishmaniasis are endemic in the Brazilian Amazon Basin. The disease occurs all over the region. To date, six species of the parasite have been isolated from different vertebrate and invertebrate hosts, including humans. Among them we have L. chagasi, L. amazonensis, L. lainsoni, L. shawi, L. naiffi, L. braziliensis and L. guyanensis (Grimaldi & Tesh, 1993. Clinical Microbiology Reviews, 6: 230).

In order to obtain information on the epidemiological features of leishmaniasis in humans in the Brazilian Amazonian Basin 65 inhabitants were thoroughly examined by clinical, parasitological and immunological (leismanin skin test) examinations. Among the patients all of them were clinically positive for dermal (89.2%), mucosal (3.1%) or mucocutaneous (7.7%) lesions. Only 37 had the skin test performed and 100% were positive. Most of the patients (81.5%) were male ranging from 20-39 years old (63.1%), acquired the disease in a rural area (80%), presented one (49.2%) or two (18.5%) lesions and some were previously treated (56.9%).

The parasites were successful isolate from all patients and were characterized by enzyme electrophoresis using nine enzymatic loci (MDH, IDH, ME, G6PDH, 6PGDH, GPI, PEPD, NH1 and NH2). Among the parasites 87.7% were characterized as L. guyanensis while 12.3% were characterized as other species (1.5% L. amazonensis; 4.6% L. braziliensis and 1.5% L. naiffi).

The genetic variability of the L. guyanensis populations is being availed using different approach. A considerable level of heterogeneity has been observed using RFLP of the rRNA ITS gene (Cupolillo et al. 1995. Mol. Biochem. Parasitol., 73: 145). The correlation of the variability and the epidemiological aspects will be discussed.

Supported by CAPES, CNPq, FIOCRUZ and INPA.

IN VIVO SELECTION OF A POPULATION OF TRYPANOSOMA CRUZI AND CLONES RESISTANT TO BENZNIDAZOLE

Murta S. M. F.^1,2& Romanha A. J.¹

¹ Laboratório de Parasitologia Celular e Molecular - Centro de Pesquisas "René Rachou" - FIOCRUZ, Caixa Postal 1743, Belo Horizonte, MG-Brazil ²Departamento de Bioquímica e Imunologia, ICB-UFMG, Brazil.

A benznidazole-resistant population of T. cruzi, Y strain, was selected after 25 successive passages (8 months) in mice treated with a single high drug dose. At the beginning of the selection, the resistant parasites produced a low parasitemia level and a low mortality rate in infected mice. Thereafter, the parasitemia level and mortality rate increased to the same value obtained for mice infected with the T. cruzi wild-type strain. Long-term treatment with benznidazole (100mg/kg/day) cured 71-80% of mice infected with wild-type strain whereas no cure was observed in mice infected with the selected resistant parasite population. Rapid treatment of infected mice with a single high dose of benznidazole (500mg/kg) at peak parasitemia cleared all blood parasites from mice infected with wild-type parasites whereas no change was observed in the parasitemia level of the selected parasites. The benznidazole-resistant parasites showed cross-resistance to Nifurtimox, Megazol and MK-436. Contrary to wild-type, all ten clones analyzed from the resistant T. cruzi population were resistant to benznidazole. The resistance phenotype of the population was stable after three passages in mice without drug pressure, whereas the resistance phenotype of the clones was stable even after 20 passages. The resistance phenotype was also stable either after 6 months of parasite maintenance in LIT medium or after one passage in Triatoma infestans. The selected population was twice as much resistant than the wild type population in culture. This work demonstrates for the first time, the in vivo selection of a population, and clones, of T. cruzi resistant to benznidazole, and thus making available an experimental model for the study of mechanisms of drug resistance in T. cruzi.

Supported by CNPq, FIOCRUZ, PAPES and PRONEX.

122

IN VIVO SELECTION OF TRYPANOSOMA CRUZI BENZNIDAZOLE MEDIUM-RESISTANT POPULATIONS FROM THE CL STRAIN AND CL BRENER CLONE HIGHLY SENSITIVE TO DRUGS

Murta, S.M.F.^1,2, Alves R.O. ² & Romanha, A.J.²

1-Departamento de Bioquímica e Imunologia- ICB-UFMG. 2- Laboratório de Parasitologia Celular e Molecular-Centro de Pesquisas "René Rachou" FIOCRUZ- Av. Augusto de Lima, 1715, 30190-002, Belo Horizonte, MG-Brazil. Email: Romanha@netra.cpqrr.fiocruz.br

The Trypanosoma cruzi CL strain and the CL Brener clone which are highly sensitive to benznidazole or nifurtimox, 90-100 % cure rates in mice, were used. To select T.cruzi resistant populations, infected mice were treated at the peak of parasitemia with 250mg of benznidazole/kg, at a single dose, orally. The treatment evaluation was made 4 h after drug administration and the remaining parasites were inoculated in a new group of mice, that was thereafter submitted to the same procedure 10 times. The number of successive treatments influenced the curve of parasitemia. Mice infected with 10 X selected parasites from CL strain and CL Brener clone produced a peak of parasitemia 8.5 and 4.5 times lower than the unselected parasites, respectively. On the 23rd day of infection the mortality rate was 100% for the mice infected with unselected parasites from CL and CL Brener clone, whereas both the 10 X selected parasites produced no mortality in the infected mice. At the 50th day the mortality produced by the selected clone was 30% and by the selected strain 0%. The selection of resistance "in vivo" was evaluated after long-term treatment (100 mg of benznidazole/kg, during 20 days, beginning 10 days after inoculation). One month after the treatment, hemocultures of mice were made and 30 days after they were examined. In the group of mice inoculated with unselected parasites from CL strain and CL Brener clone the percentage of cure was 90 and 92%, whereas the group of mice infected with the CL strain and CL Brener clone selected 10 X the cure was 22 and 40%, respectively. A reduction of 100% in the parasitemia was observed in the mice infected with unselected parasites from T. cruzi CL strain and CL Brener clone, treated with a single 250 mg/kg dose of benznidazole after 6 hr of rapid treatment. In contrast, a reduction of 39 and 62% in the parasitemia was observed in the infected mice with 10X selected parasites from CL strain and CL Brener clone, respectively. Similar reductions in parasitemia were observed when the mice were treated with a single dose of 500 mg/kg. The results demonstrate the in vivo selection of a benznidazole medium-resistant population from CL strain and CL Brener clone of T.cruzi. Compared with the original strain, the population of selected parasites produced in inoculated mice a lower parasitemia, no mortality and a lower percentage of cure after the long-term treatment.

Supported by CNPq, PAPES/ FIOCRUZ and PRONEX.

123

CHAGAS' DISEASE AND HIV CO-INFECTION: BIOLOGICAL BEHAVIOUR OF TWO TRYPANOSOMA CRUZI STRAINS

Brito, C.M.M ¹ ; Chicarino, J.M.C ² ; Da Cruz, A. M³ ; Machado, M. I ⁴ & Pacheco, R. S ⁵.

¹Departamento de Ciências Biológicas, ENSP; ²Setor de Anatomia Patológica-Hospital Evandro Chagas ; ³Departamento de Protozoologia ; ⁵Departamento de Bioquímica e Biologia Molecular, IOC-FIOCRUZ. ⁴Universidade Federal de Uberlândia.

We have previously shown that T..cruzi strains isolated from HIV positive patients demonstrated low parasitaemia and low virulence patterns in imunocompetent mice. However the literature has reported cases of reactivation of Chagas'disease in association with AIDS, following by the involvement of the central nervous system (CNS). Aiming to evaluate the role that specific parasite populations play in the severity of clinical manifestations, we have been investigating the behaviour of these strains in the C3H, Balb/C and albino mice during acute, chronic phase and also under immunosupression condition (chronic phase of disease). To improved the parasite virulence , the organisms were maintained under different laboratory conditions by in vitro culture (group 2) and serial passage in mice (group 1). Different genotypic profiles were detected in those groups suggesting polyclonality of the primary isolate (patient) and a possible selection of specific subpopulations.

Our results allow us to conclude that (i) reactivation of the disease occured in 50% of the mice in chronic phase (35/70) after immunosupression with cyclophosphamide, (ii) high parasitaemia levels ( parasitemic peaks between 22 and 30th day ) were observed in C3H following by albino and Balb/c mice when inoculated with parasites from prolonged in vitro and in vivo maintenance, (iii) absence of mortality in mice was observed with one strain. With the other one the mortality rate was around 9,5% (2/21 albino and C3H mice), (iv) histopathological examinations showed myocarditis without amastigotes and no involvement of the CNS, (v) severe myocarditis with amastigotes and meningoencephalitis could be observed in mice inoculated with parasites after prolonged maintenance.

Our results also suggest the possibility of selection with predominance of a parasite subpopulation with characteristics of low patogenicity. Work is in progress regarding clonal analysis in order to define the population structure of these strains.

Supported by International Atomic Energy Agency (IAEA - Vienna / Austria).

124

MORPHOLOGICAL DIFFERENCES AMONG DIFFERENT STRAINS AND CLONES OF TRYPANOSOMA CRUZI

Nascimento, H.F. & Souto-Padrón, T.

Laboratório de Protozoologia I , Instituto de Biofísica Carlos Chagas Filho - UFRJ - CCS, Bloco G - Ilha do Fundão, Rio de Janeiro, RJ, 21949-900 - BRASIL.

The general structure of Trypanosomatids has been subject of several studies (De Souza, Int.Rev.Cytol. 86:197,1984). Cell surface of those protozoan are composed by the cell coat, the plasma membrane and an organized layer of microtubules located immediately below the plasma membrane called sub-pellicular microtubulles that underline the whole protozoan body, excepting the region of flagellar pocket. Until now morphological studies comparing the sub-pellicular microtubules in some Trypanosomatids indicated structural differences. In T. cruzi the sub-pellicular layer impairs the approach of organelles to the plasma membrane while in Leishmania smooth endoplasmic reticulum tubules can pass through this layer to stay very close to the plasma membrane. Trypanosoma cruzi however, is not an homogeneous population and it is composed by a pool of strains showing distinct characteristics. This intraspecific variation reflects differences concerning: curves of parasitemia,virulence, pathogenicity,sensitivity to drugs,presence of strain specific antigens, different membrane carbohydrate residues, induction of capping and shedding, zymodeme, schizodeme and nuclear DNA diversity showed by molecular karyotiping,DNA fingerprinting and RAPD analysis. Recently, Zingales et al (Mem. Inst. Oswaldo Cruz 91 Suppl. 14-15,1996) presented several data showing a division of T. cruzi into two groups or lineages that differ at the DNA level.

In this study we analysed the ultrastructure of epimastigote forms of the Y and CL strains and CL14 clone (lineage 1) and Colombiana strain and Dm28c clone (lineage 2) cultivated in LIT medium. Parasites were fixed in 2.5 % glutaraldehyde, 4% formaldehyde in 0.1 M cacodylate buffer containing 3.7 % sucrose during 1 h at room temperature. Samples were post-fixed in 1% osmium tetroxyde, dehydrated in acetone and embedded in Epon. Thin sections were analysed in ZEISS 900 transmission electron microscope. Sub-pellicular microtubule layer of Colombiana and Dm28c epimastigotes presented a different pattern of distribution when compare to the observed in Y and CL strains and CL14 clone presenting the same morphological characteristic described for Leishmania. Moreover we also observed in Dm28c clone a distinct paraflagellar structure. A deep analysis is going on in order to investigate other strains bellonging to both different T. cruzi lineages.

Supported by PRONEX, FINEP, CNPq & CEPG-UFRJ

125

ISOENZYMATIC CHARACTERIZATION OF ISOLATES OF T. EVANSI FROM THE PANTANAL IN MATO GROSSO STATE, BRAZIL.

¹Queiroz, A.O.; ¹Corrêa, R.P.G.; ¹Menezes, V.T.; ³D'Avila, A.M.R.; ³ Silva, R.A.M.S.;² Leon, L.L. and ¹ Jansen, A.M.

Departamento de Protozoologia, ¹Laboratório de Biologia de Tripanosomatídeos, Instituto Oswaldo Cruz/FIOCRUZ ²Laboratório de Bioquímica de Tripanosomatídeos, Instituto Oswaldo Cruz/FIOCRUZ ³EMBRAPA/Mato Grosso do Sul.

Av.Brasil 4365, Manguinhos,21045-900, Rio de Janeiro,RJ, Brasil

Trypanosoma evansi, a flagellated kinetoplastid of the family Trypanosomatidae is classified within the subdivision Salivaria (Hoare, 1972) according to its mode of inoculation: mechanical transmission via hematophagic insects of the geni Tabanus and Stomoxys. In Latin America this parasite has acquired an additional vector, the bat Desmodus rotundus. In Brazil, T. evansi is responsible for an equine disease known as "Mal de Cadeiras", which causes ataxia in horses, thus limiting their use in raising cattle in open areas in the livestock-dependent regions of the Pantanal in Mato Grosso State and the Ilha de Marajó (Pará State).

We examined six isolates which were derived from the following hosts: 2 isolates from horse hosts; 2 isolates from dogs; and 2 isolates from quati(Nasua nasua, carnivora, Procyonidae) which were infected by natural means in the Pantanal of Mato Grosso State. These specimens were cryopreserved in our laboratory. Rats which had been immunosuppressed with 200mg/kg of cyclophosphamide 48 hours prior, were inocculated with the tripomastigote forms of T. evansi. Blood was drawn from these inocculated rats and the parasites purified using anion-exchange chromatography on a DEAE-cellulose column ( Lanham and Godfrey, 1970). The purified parasites were lysed and the enzymatic activity of a certain extract was analyzed in an attempt to differentiate among the six isolates using substrates of the following enzymes: MDH [EC 1.1.1.37 ],G6PI [EC 5.3.1.9], G6PDH [EC 1.1.1.44], IDH [EC 1.1.1.42] and ME [EC 1.1.1.40]. It was observed that all of the isolates demonstrated similar electrophoretic profiles. The homogeneity of isoenzymatic profiles of T. evansi had already been observed by other authors in isolates derived from domestic animals and capybaras(Stevens,1989; Franke,1994). Additionally, our results show that isolates from quati have the same profile. T. evansi has one of the widest distributions and one of the greatest range of mammalian hosts, and it is agreed that it evolved from T. brucei. This adaptability is not reflected in the variability of the biochemical parameters studied until now (although we observed significant variability in the virulence for Swiss mice). An interesting aspect of this parasite is that T. evansi was introduced in South America by domestic animals during the Spanish colonization, after which it adapted to wild animals and established a sylvatic transmission cycle.

126

HERPETOMONAS FROM PLANTS AND PHYTOPHAGOUS HEMIPTERANS: ARE SOME HERPETOMONAS spp. HETEROXENEOUS?

Sousa, M.A.⁽¹⁾, Tavares, C.C.⁽²⁾, Fiorini, J.E⁽³⁾, Sá-Xavier, C.^* * (CNPq , FAPERJ ** , FAPEMIG *** Fellowships) (1), Campêlo, A.G^** * (CNPq , FAPERJ ** , FAPEMIG *** Fellowships) (1), Santos, S.M.⁽¹⁾ & Cucolichio,G.^*** * (CNPq , FAPERJ ** , FAPEMIG *** Fellowships) (3)

(1) - Coleção de Tripanosomatídeos, Depto Protozoologia, Instituto Oswaldo Cruz, CP 926 - 20001-970 Rio de Janeiro, RJ; (2) Depto Biologia Celular e Genética, Instituto de Biologia, Universidade do Estado do Rio de Janeiro; (3) Depto Ciências Biológicas, UNIFENAS, Alfenas, MG.

Fiorini et al. isolated from a tomato fruit and a hemipteran Phthia picta captured on it, as well as from Leptoglossus stigma found in association with oranges, trypanosomatids which were identified as Phytomonas (Cytobios 75: 163-170, 1993; Mem. Inst. Oswaldo Cruz, 85 suppl: 134, 1990). These strains were deposited in the Trypanosomatid Collection at the Oswaldo Cruz Institute under the code number CT-IOC 229, 228 and 221, respectively. They were indistinguishable from each other when examined in Giemsa-stained smears, but considerably differed from six isolates of tomatoes and phytophagous hemipterans obtained by Jankevicius & Itow-Jankevicius, all identified as Phytomonas serpens.

These strains were comparatively analyzed by pulsed-field gel electrophoresis (PFGE) under different running conditions and by molecular hybridization using b-tubulin gene probes. The growth and cellular differentiation in LIT medium at 27.3^oC was also compared in the isolates obtained by Fiorini et al. and in two P.serpens strains (9T and 1G). Two Herpetomonas species from insects (H. megaseliae and H. muscarum muscarum) and two others from plants (H. davidi and the isolate of Euphorbia hyssopifolia obtained by Attias & De Souza 1986) were included in the present study as references. We evidenced that the isolates obtained by Fiorini et al. in fact belonged to the Herpetomonas genus, since they presented paramastigotes and opisthomastigotes (although at low rates) in their cultures; these stages were not found in the organisms identified as P. serpens. The isolates obtained by Fiorini et al. presented identical DNA banding pattern by PFGE, as well as the same localization of b-tubulin genes, both being similar to those presented by the Herpetomonas species, this confirming their generic identification. The six P. serpens strains were similar to each other and also similar to a new isolate obtained from tomato and Phthia picta insects from Alfenas (MG), all being clearly distinct from the Herpetomonas species.

Our results evidence that a same trypanosomatid belonging to the Herpetomonas genus can be isolated both from a plant and phytophagous insects. The question which arises is whether at least some Herpetomonas species can be heteroxeneous. It is worthy mentioning that the so-called "Phytomonas" davidi was experimentally transmitted from plant to plant through the hemipteran Pachybrachius bilobata scutellatus (McGhee & Postell 1982), that parasite possibly being the H. davidi. Interestingly, another trypanosomatid isolated in axenic culture from Phthia picta (salivary glands) was also identified as a Herpetomonas sp. (Sousa et al. 1995).

127

COMPARATIVE STUDY OF SECRETED PHOSPHATASE ACTIVITY BETWEEN TRYPANOSOMATIDS PARASITES OF PLANTS AND OF HOUSE FLIES.

Dutra, P.M.L.¹, Santos, M.A.A.¹, Couto, L.C.¹, Rodrigues, C.O.¹, Barros, F.S.¹, Lopes, A.H.C.S.¹, Romeiro, A.³, Attias, M.³ & Meyer-Fernandes, J.R.²

¹Instituto e Microbiologia Prof. Paulo de Góes, ²Departamento de Bioquímica Médica, I.C.B., ³Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21941-590 Rio de Janeiro, R.J., Brasil.

Studies carried out in the last 21 years have pointed out the wide presence of trypanosomatids in different plants, some of them of large economical importance, such as coconut (Parthasarathy et al., 1976 Science 192 : 1346-1348), oil palm (Van Slobe et al., 1978. J. Palm. Soc. 22 : 25), tomato (Jankevicius et al., 1989. J. Protozool. 36 : 265-271) and corn (Jankevicius et al., 1993. J. Euk. Microbiol. 40: 576-581). In some cases, evidences were obtained showing the involvement of insects as intermediary hosts and vectors of such parasites (Sbravate et al., 1989. J. Protozool. 36 : 534-547; Brazil et al., 1990. Mem. Inst. Oswaldo Cruz 85 : 239-240). Phosphatase activity has been characterized in some members of the family Trypanosomatidae, such as Leishmania and Trypanosoma, which are pathogenic for humans and other mammals. In this work, we have characterized a secreted phosphatase of two trypanosomatids parasites of plants, Phytomonas françai and Phytomonas serpens, and one parasite of insect, Herpetomonas muscarum muscarum, which infects house flies. The parasites were grown in Warren medium for 4 days at 28°C and incubated in Tris-HCl (100 mM)/sucrose (250 mM) pH 6.8. The supernatants were used to detect the phosphatase activity and they were shown to hydrolized p-nitrophenylphosphate (p-NPP) a rate of 15.15 nmol Pi/mg . min, for P. françai, 10.26 nmol Pi/mg . min, for H. m. muscarum and 6.51 nmol Pi/mg . min, for P. serpens. The three secreted phosphatases presented the same pattern in response to variations of pH, where the optimum activity was found to be at pH 5.5. These activities shown a hyperbolic dependence of substrate concentration, where the aproximated K_m found was 1.96 mM p-NPP for H. m. muscarum, 1.46 mM p-NPP for P. serpens and 2.63 mM p-NPP for P. françai. Sodium tartrate, a known inhibitor of acid phosphatase secreted by some species of Leishmania, was able to inhibit the secreted phosphatase activities. Experiments using classical inhibitors of acid phosphatase, such as sodium orthovanadate, sodium fluoride and amonium molybdate, promoted a decrease in these enzymes activities.

Supported by: FINEP, CNPq and PRONEX (41/96 0885.00)

128

CROSS-REACTIVITY BETWEEN LEISHMANIA AND TRIPANOSOMATIDS ISOLATED FROM PLANTS AND A PHYTOPHAGOUS INSECT (TIPPI) BY WESTERN BLOTTING (WB).

Costacurta, R.A.; Oliveira, F.L. de; Rocha, K.M.; Yang, A.V.; Tano, M.S.; Reiche, E.M.V.; Gonçalves, C.C.M.; Okamura, H.; Itow-Jankevicius, S.; Jankevicius, J.V. Departamento de Patologia Geral, Centro de Ciências Biológicas, Universidade Estadual de Londrina - Caixa Postal 6001, CEP 860510-900, Londrina-PR.

The preparation of antigens for American Cutaneous Leishmaniasis (ACL) diagnosis is generally derived from Leishmania cultures, it is expensive , easily contaminated and potentially infectious for laboratory workers (Terry et al., 1950; Sampaio et al., 1983; Dillon et al., 1993). The presence of cross-reactivity between genus Leishmania and TIPPI recognized in DAT (Okamura et al., 1995) gave us rise to try better characterization of the shared antigens through WB technique. L.amazonensis (MHOM/BR/89/M12766) and L.tropica (ATCC 30012) strains were cultivated in blood agar slants (Evans et al., 1984) with an overlay of LIT (Camargo, 1964) at 24°C. Phytomonas serpens (Jankevicius et al., 1987), 415Ga (isolated from salivary gland of Leptoglossus sp in Londrina/PR, 1990), 268Tb (Almeida, 1993) and Herpetomonas mcgheei (Itow-Jankevicius et al., 1993) cultivated in blood agar/GYPMI (Itow-Jankevicius et al., 1993) at 28°C were also used. Protein was extracted from cultures in lag, log and stationary phases by ultrasonication (Reiche, 1996) or by chemical lysis (modified from Maizels, 1991), with storage at -20°C or 4°C. The SDS-Polyacrilamide Gel Electroforesis (SDS-PAGE) (Laemmli, 1970) was carried out in a gradient varying from 7.5 to 15%. Gels were submitted to densitometry or transfered to nitrocellulose membranes and WB (Towbin et al., 1979) with human sera. These sera were ACL+ (Indirect Immunofluorescence Test), ACL- or from patients with other pathologies. Preliminary results demonstrated loss (qualitative and/or quantitative) of certain protein bands in mechanic procedure when compared with chemical lysis. Qualitative or quantitative differences of some band protein profiles of lisates obtained from different growth phases of the TIPPI strains. There was no differences in eletroforetic profiles of strains in relation to the methodology of antigen storage.

Financial support: CPG-UEL, CAPES and CNPq

e-meil: okamuray@sercomtel.com.br

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