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The Brazilian National Biosafety Committee approved in 2011 a new post release monitoring system for environmental releases of genetically modified organisms. It has a number of novel features in comparison with other established or proposed systems. The new system also allows the proponent to ask for monitoring exemption. General surveillance forms the basis of the monitoring system, similar to the European model, but differs markedly in the way it operates. While the European proposal is based on monitoring measurable variables extracted from environmental observations, from baselines previously established for multiple protection targets, the Brazilian system uses direct alerts of damage, without the aid of baseline values. The strength of the Brazilian form of monitoring is the possibility of generating an information network with the effective participation of many actors from the monitored area. A network constituted by highly qualified members, as proposed elsewhere, is too complex and unrealistic in Brazil and in many other countries. In conclusion, the Brazilian monitoring system is flexible and can be adjusted to the Brazilian reality over the next years, as a response to the ever growing experience in monitoring. It also meets the demands of the Brazilian society for transparency, rational use of resources, opportunity for national companies, and food and environmental biosafety.

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Serodiagnosis of visceral leishmaniasis is often hindered by cross-reactions to other parasitic diseases. Identifying specific B-cell epitopes in proteins is therefore important for immunodiagnostics, as well as for disease control by vaccines. This study aimed to identify linear and conformational B-cell epitopes and to evaluate the secondary structure of antigen proteins in Leishmania infantum using in silico analysis. Linear epitopes were predicted using the Immune Epitope Database and Analysis Resource (IEDB), BepiPred and BcePred programs. The conformational B-cell epitopes were identified using the CBTOPE server. The combination of the predictions using IEDB, BepiPred and BcePred generated 148 linear epitopes from the calpain-like cysteine peptidase (CP), thiol-dependent reductase 1 (TDR1) and HSP70 proteins. In total, 164 conformational epitopes were predicted, mostly located in the linear epitope region. The predicted epitopes are located in α helix and random coil regions in the thiol-dependent reductase 1 and HSP70 proteins. New linear and conformational B-cell epitopes of L. infantum proteins were identified in silico, and the prediction using various programs ensures greater accuracy of the results, as suggested by confirmation of previously identified HSP70 epitopes.

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Defensin, thionin and lipid transfer protein (LTP) gene families, which antimicrobial activity has an attractive use in protein engineering and transgenic production of agronomical important plants, have been here functionally reviewed. Also, a transcriptional overview of a set of plant SuperSAGE libraries and analysis looking for 26 bp tags possibly annotated for those families is presented. Tags differentially expressed (p = 0.05) or constitutively transcribed were identified from leaves or roots SuperSAGE libraries from important Brazilian plant species [cowpea (Vigna unguiculata (L.) Walp.), soybean (Glycine max (L.) Merr.) and modern sugarcane hybrids (Saccharum spp.)] submitted to abiotic [salt (100 mM NaCl) or drought] or biotic stresses [fungus inoculation (Phakopsora pachyrhizi; Asiatic Soyben Rust phytopathogen)]. The diverse transcriptional patterns observed, probably related to the variable range of targets and functions involved, could be the first step to unravel the antimicrobial peptide world and the plant stress response relationship. Moreover, SuperSAGE opens the opportunity to find some SNPs or even rare transcript that could be important on plant stress resistance mechanisms. Putative defensin or LTP identified by SuperSAGE following a specific plant treatment or physiological condition could be useful for future use in genetic improvement of plants.

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ABSTRACT The serodiagnosis of small ruminant lentivirus (SRLV) infections in goats and sheep is usually performed by the agar-gel immunodiffusion technique (AGID) and by ELISA. Therefore, the western blot (WB) is the choice for the definition of antigen recognition patterns during the disease progression, being potentially useful for the diagnosis itself. In the present study, the antigen used for WB was obtained by a novel simple purification procedure: the dialysis of the supernatant of infected cultured cells, followed by centrifugation in sucrose gradient. Proteins in the pellet were separated by SDS-PAGE at 10% density and transferred to nitrocellulose membranes in a semi-dry blotting device. After development, five viral proteins were recognized by the standard positive serum sample, with molecular weights of 14-16, 25, 40, 50 and 70 kDa respectively. All 8 AGID positive goat serum samples recognized at least one band in the immunoblot, with different intensities. The total number of bands recognized by each serum sample varied considerably. A positive reaction to gp40 was observed with 4 sera, although rather weak in 3 cases. Two samples were reactive to p16 and two others to gp50, although weakly. Curiously, no serum was reactive to the 70 kDa antigen, recognized by the standard positive serum sample. These results suggest that the WB can be effectively used for the routine serodiagnosis of viral caprine arthritis encephalitis infection (CAEV). It can also support further studies on antigen recognition patterns with the new antigen provided by the novel simplified purification system of viral components described here.

RESUMO O sorodiagnóstico das lentiviroses de caprinos e ovinos é realizado principalmente pela imunodifusão em gel de agar (AGID) e/ou ELISA. Embora relativamente simples, esses métodos não identificam os antígenos virais reconhecidos na resposta imune do animal examinado, por isso o western blot (WB) vem ganhando maior relevância como ferramenta de diagnóstico dessas enfermidades. Neste trabalho, o antígeno utilizado no WB foi obtido através de um sistema simplificado de purificação: concentração por diálise do sobrenadante de culturas celulares infectadas, seguido de centrifugação em gradiente contínuo de sacarose. A separação das proteínas virais foi obtida por SDS-PAGE a 10% e a transferência para membranas de nitrocelulose realizada pelo sistema semi-úmido. A revelação das membranas mostrou reconhecimento pelo soro padrão positivo de cinco proteínas, com pesos moleculares de 14-16, 25, 40, 50 e 70 kDa. Todas as 8 amostras de soro caprino, positivas na ADIG, reconheceram pelo menos uma banda proteíca no immunoblot, variando contudo o número de bandas reconhecidas. Reação positiva à glícoproteína 40 (gp 40) foi observada em quatro animais, com intensidade de reação discreta em três deles. Dois animais apresentaram reação positiva à proteína 16 (p16), e dois à gp 50, de pouca intensidade. Quanto à gp 70, proteína que, embora reagisse com o soro padrão positivo, não foi reconhecida por nenhum dos soros testados. Estes resultados sugerem que o WB pode ser empregado para o sorodiagnóstico rotineiro das lentiviroses, ensejando estudos mais amplos do padrão de reconhecimento dos antígenos apresentados por este novo sistema de purificação parcial de componentes virais.

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Chemotherapy for Chagas' disease is still unsatisfactory due to toxicity and limited effectiveness of the available drugs. In this work we have investigated the effect of usnic acid, isolated from lichen Cladonia substellata, against Trypanosoma cruzi, in vitro. Incubation of culture epimastigotes with 5-30microg/ml of this compound resulted in growth inhibition in a dosis-dependent manner. Ultrastructural analysis of treated epimastigotes showed damage to mitochondria, with a marked increase in kinetoplast volume and vacuolation of the mitochondrial matrix. Intense lysis of bloodstream trypomastigotes was observed with all drug concentrations tested. Besides mitochondrial and kinetoplast damage, trypomastigotes also presented enlargement of the flagellar pocket, as well as intense cytoplasm vacuolation. Treatment of infected macrophages with 40 or 80microg/ml usnic acid induced marked cytoplasm vacuolation in intracellular amastigote forms, with disorganization of parasite kinetoplast and mitochondria, but with no significant ultrastructural damage to the host cells.

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Lutzomyia umbratilis, a known vector of Leishmania guyanensis in the north of Amazon basin, has been exclusively found in the Amazonian region. Here we report for the first time the occurrence of this species in northeastern Brazil. The epidemiological importance of the occurrence of this species in the Atlantic Forest is commented.

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Using transmission electron microscopy, gold-labeled lectins, morphometry and enzyme-linked lectin assay, we could show that treatment of promastigotes of Leishmania donovani chagasi with trypsin did not interfere with the binding of lectins (concanavalin A, peanut agglutinin, wheat germ agglutinin and Ricinus communis agglutinin) to the parasite surface. These observations are in agreement with results we previously obtained using a biochemical approach. Treatment of fixed promastigotes with 2-mercaptoethanol induced a significant increase in the density of concanavalin A (Con A) receptors on the surface of L. d. chagasi in relation to the control. We suggest that this increase is due to the unfolding of one or more surface glycoproteins after cleavage of disulfide bonds between cystein residues in adjacent protein loops, exposing second-order Con A receptors that are otherwise hidden in the protein quaternary structure.

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Crithidia fasciculata is an important trypanosomatid parasite commonly affecting insects and is used extensively as a model for the study of the biochemistry, ultrastructure and organization of the kDNA network of trypanosomatids. The present study describes the evolution of UV-induced morphological changes detectable by transmission electron microscopy in Crithidia fasciculata. Although only rare and minor changes in kinetoplast DNA were demonstrable 7 h after UV irradiation, alterations of this organelle were present in almost all flagellates observed 24 h and 48 h after irradiation. Other cell structures were apparently undamaged. Ultrastructural changes in kDNA did not correspond to changes in antigenicity of protein bands in western blotting against serum from Chagas' disease patients or in the presence of 3 different lectin receptors on the surface of the parasite.

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The need for a reliable method for the immunological diagnosis of kala-azar is imperative. Leishmania donovani donovani and L. donovani chagasi culture promastigotes were compared as antigens in a direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis in Brazil. Both antigens were successfully employed for the DAT, showing 100% sensitivity and greater than 98% specificity when used to test sera from Brazilian and African kala-azar, Chagas' disease, malaria, filaria and syphilis patients, and on sera from Brazilian controls. Cross-reactions were sometimes observed when cutaneous and mucocutaneous leishmaniasis patient sera were tested. The cross-reactions were completely abolished by the addition of 0.78% 2-mercaptoethanol to the serum diluent. These data show that this improved DAT can be used for the diagnosis of visceral leishmaniasis in Brazil.

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The need for a reliable method for the immunological diagnosis of Kala-azar is imperative. Leishmania donovani donovani and L. donovani chagasi culture promastigotes were compared as antigens in a direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis in Brazil. Both antigens were successfully employed for the DAT, Showing 100½ sensitivity and > 98% specificity when used to test sera from Brasilian and African Kala-azar, Chagas'disease, malaria, filaria and symphilis patients, and on sera from Brazilian controls. Cross-reactions were sometimes observed when cutaneous and cucocutaneous leishmaniasis patient sera were teste. The vross-reactions were completely abolished by the addition of 0.78% 2-mercaptoethanol to the serum diluent. These data show data that this improved Dat can be used for the diagnosis of visceral leishmaniasis in Brazil

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Crithidia fasciculata is an important trypanosomatid parasite commonly affecting insects and is used extensively as a model for the study of the biochemistry, ultrastructure and organization of the kDNA network of trypanosomatids. The present study describes the evolution of UV-induced morphological changes detectable by transmission electron microscopy in Crithidia fasciculata. Although only rare and minor changes in Kinetoplast DNA were demonstrable 7 h after UV irradiation, alterations of this orgtanelle were present in almost al flagellates observed 24 h and 48 h after irradiation. Other cell structures were apparently undamaged. Ultrastructural changes in kDNA did not correspond to changes in antigenicity of protein bands in western blotting against serum from Chagas' disease patients or in the presence of 3 different lectin receptors on the surface of the parasite

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The effects of short ultraviolet (253 mm) radiation on the growth of the trypanosomatid flagellate Crithidia fasciculada were studied in liquid medium and nutrient agar plates. Cell duplication was completely inhibited after exposure of the flagellates to doses equal to or higher than 50 J/m2. The UV-induced lag period was dose-dependent. Survival was reduced to 1% after exposure of the parasites to 200 J/m2. Ultrastructural changes after the lag period were studied by transmission electron microscopy. Changes of the kinetoplast network structure and sometimes of the mitochondrial matrix were observed. The existence of DNA repair mechanisms in this protozoan is discussed

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An enzyme-linked lectin assay (ELLA) based on the ELISA assay, using intact formalin-fixed promastigotes to coat poly-L-lysine-treated microtiter plates is described. The assay was used to study the lectin receptors of Leishmania donovani chagasi, L. donovani donovani and L. mexicana amazonensis. ConA, RCA, WGA, and PNA receptors were found in the three parasites. SBA receptors were found to be as frequent as the other receptors in L. donovani chagasi but not in the other two parasites which showed little SBA binding. Trypsin treatment of the two L. donovani subspecies did not remove any of the lectin receptors studied

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L. donovani antigens were analyzed by a direct agglutination test (DAT), by ELISA using intact promastigotes and by the SDS-PAGE immunoperoxidase technique (SGIP). Sera of Chagas' disease patients cron-reacted in the ELISA and SGIP but not in the DAT. Trypsin treatment of the parasites removed concanavalin A-binding sites but not epitopes for antibodies present in Chagas' disease and in leishmaniasis sera, as seen in the SGIP. Eight bands were revealed after incubation of the gel sections with Kala-azar or Chagas' disease sera, three of which were common to both sera. The major antigenic component recognized by leishmaniasis sera was a glycoprotein of 57 KD, and the major cross-reacting protein recognized by Chagas' disease sera was a glycoprotein doublet of 71.5-68KD

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L. donovani antigens were analyzed by a direct agglutination test (DAT), by ELISA using intact promastigotes and by the SDS-PAGE immunoperoxidase technique (SGIP). Sera of Chagas' disease patients cross-reacted in the ELISA and SGIP but not in the DAT. Trypsin treatment of the parasites removed concanavalin A-binding sites but not epitopes for antibodies present in Chagas' disease and in leishmaniasis sera, as seen in the SGIP. Eight bands were revealed after incubation of the gel sections with kala-azar or Chagas' disease sera, three of which were common to both sera. The major antigenic component recognized by leishmaniasis sera was a glycoprotein of 57 kD, and the major cross-reacting protein recognized by Chagas' disease sera was a glycoprotein doublet of 71.5-68 kD.

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Herpetomonas samuelpessoai was submitted to hypo- and hyper-osmotic shocks in buffer and culture medium. Cell volume was evaluated with an electronic particle counter and by the transmission of a parasite suspension measured at 540 nm with a spectrophotometer. Cell volume changes after osmotic shock were transient, parasites being able to completely restore cell volume to the original values in culture medium but not in buffer. The kinetics of transmittance changes closely resembled that of volume variations. The volume regulatory increase (but not decrease) was dependent on extracellular K concentration and strongly influenced by the parasite growth phase. Evidence is presented supporting the idea that changes in transmission values can be linearly correlated to mean cell volume variations in free-floating cells.

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The effects of short ultraviolet (253 nm) radiation on the growth of the trypanosomatid flagellate Crithidia fasciculata were studied in liquid medium and nutrient agar plates. Cell duplication was completely inhibited after exposure of the flagellates to doses equal to or higher than 50 J/m2. The UV-induced lag period was dose-dependent. Survival was reduced to 1% after exposure of the parasites to 200 J/m2. Ultrastructural changes after the lag period were studied by transmission electron microscopy. Changes of the kinetoplast network structure and sometimes of the mitochondrial matrix were observed. The existence of DNA repair mechanisms in this protozoan is discussed.

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An enzyme-linked lectin assay (ELLA) based on the ELISA assay, using intact formalin-fixed promastigotes to coat poly-L-lysine-treated microtiter plates is described. The assay was used to study the lectin receptors of Leishmania donovani chagasi, L. donovani donovani and L. mexicana amazonensis. ConA, RCA, WGA, and PNA receptors were found in the three parasites. SBA receptors were found to be as frequent as the other receptors in L. donovani chagasi but not in the other two parasites which showed little SBA binding. Trypsin treatment of the two L. donovani subspecies did not remove any of the lectin receptors studied.

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Two-hundred and sixty-five human sera from Brazil were tested in a direct agglutination assay using trypsinated Leishmania donovani donovani antigen. The assay proved to be of value for the diagnosis of American visceral leishmaniasis in that it was discriminative toward Chagas' disease.

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