Preliminary observations of the urbanization and domiciliation of the American cutaneous leishmaniasis in Rio Branco, Acre, Western Amazon

ABSTRACT Background: The American cutaneous leishmaniasis (ACL) is expanding in peri-urban environments. Methods: An entomological survey was conducted in the area of the occurrence of an autochthonous urban case of ACL. Sandflies and a parasitological slide of the human case were submitted for molecular diagnosis. Results: Nyssomyia whitmani and Ny. antunesi were the most frequently collected species. Ny. whitmani and Bichromomyia flaviscutellata were positive for Leishmania guyanensis and L. lainsoni, respectively. The human case tested positive for L. lainsoni. Conclusions: Sandflies and Leishmania parasites present in urban forest may occur frequently in nearby domiciliary environments; thus, these areas must be monitored.

Initial survey on the use of animals in scientific research and teaching reveals divided opinion of the Brazilian population / Investigação inicial sobre o uso de animais na pesquisa científica e no ensino revela opinião dividida da população brasileira

ABSTRACT Objective: Specific legislation regulating the use of animals in research in Brazil was introduced in 2008. However, the viewpoint of the Brazilian population regarding the use of animals in research and teaching activities remains largely unknown. Investigation of the public viewpoint on and understanding of the topic is required given the current shifts in the animal ethics scenario in Brazil. The objective of this study was to provide the first insight into the Brazilian population viewpoint on the use of animals in scientific research and teaching activities. Methods: Data collected in a survey involving 2,115 individuals aged 16 years or older and residing in 130 municipalities distributed across the five Brazilian macroregions (North, Northeast, South, Southeast, and Midwest) were analyzed. The margin of error for entire sample was set at 2%, with a 95% confidence interval. Results: This survey revealed that most Brazilian citizens are in favor of the use animals in research, particularly for medical purposes. Different views depending on the nature of research were identified. Approximately 80% of respondents were also in favor of frequent oversight of laboratories and animal facilities. Conclusion: Survey findings indicate that the opinion of the Brazilian population is divided when it comes to the use of animals in scientific research and teaching. Divided opinions expose a limited understanding of the importance of basic sciences and emphasizes the need for improved communication between the scientific community and the general population. Further strategies aimed to promote animal welfare are discussed.

RESUMO Objetivo: A legislação específica que regula o uso de animais em pesquisa no Brasil foi introduzida em 2008. No entanto, a opinião da população brasileira sobre o uso de animais em atividades de pesquisa e ensino ainda é desconhecida. No atual cenário brasileiro em mudança com relação à ética animal, é necessário avaliar as visões e o conhecimento da população sobre o assunto. O objetivo deste destudo foi realizar o primeiro levantamento da opinião da população brasileira sobre o uso de animais em atividades de ensino e pesquisa científica. Métodos: Analisamos os resultados de uma pesquisa com 2.115 indivíduos com 16 anos ou mais de 130 municípios das cinco macrorregiões brasileiras (Norte, Nordeste, Sul, Sudeste e Centro-Oeste). A margem de erro para toda a amostra foi de 2% dentro de um intervalo de confiança de 95%. Resultados: A pesquisa revelou que a maioria da população brasileira era favorável ao uso de animais em pesquisas, principalmente para fins médicos. Diferentes pontos de vista, dependendo da natureza da pesquisa, também foram identificados. Além disso, aproximadamente 80% dos entrevistados eram favoráveis ao monitoramento frequente de laboratórios e instalações de animais. Conclusão: A opinião da população brasileira está dividida com relação ao uso de animais em pesquisa e ensino científicos. Essa divisão expõe um entendimento limitado da importância das ciências básicas e destaca a necessidade de uma melhor comunicação entre a comunidade científica e a população em geral. Outras ações para alcançar as melhorias desejadas no bem-estar animal são discutidas.

Detección molecular de Leishmania (Viannia) lainsoni Silveira, Shaw, Braga & Ishikawa, 1987 (Kinetoplastida: Trypanosomatidae) en humanos de Paraguay

RESUMEN La leishmaniasis es una de las enfermedades tropicales más desatendidas, causada por el parásito Leishmania. En Paraguay, la especie responsable de la leishmaniasis cutánea es (LC) es L. (Viannia) braziliensis. Aquí se reporta un caso diagnosticado de Leishmaniasis, y análisis moleculares empleando reacción en cadena de la polimerasa - polimorfismos de restricción de fragmentos de restricción (PCR-RFLP) demostraron que el caso fue causado por L. (V.) lainsoni. Este es el primer registro de esta especie para Paraguay, con lo cual se extiende el rango de distribución conocido de la especie, unos 1.430 km al sur de localidades previamente conocidas. Se necesitan más estudios para conocer la incidencia real de esta especie en casos de LC en Paraguay, y para identificar reservorios naturales del parásito en la naturaleza.

ABSTRACT Leishmaniasis is one of the most neglected tropical diseases worldwide caused by the parasite Leishmania. In Paraguay the species responsible for cutaneous leishmaniasis (CL) is L. (Viannia) braziliensis. Here we report a case diagnosed with Leishmaniasis, and molecular analyses using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) demonstrate that the case was caused by L. (V.) lainsoni. This is the first record of this species for Paraguay, with which we extend the distribution range of the parasite 1,430 km southwards from the southernmost previous known locality. More studies are needed to know the actual incidence of this species in cases of CL in Paraguay, and to identify natural reservoirs in the wild.

Identification of SL addition trans-splicing acceptor sites in the internal transcribed spacer I region of pre-rRNA in Leishmania (Leishmania) amazonensis

Trypanosomatidae is a family of early branching eukaryotes harbouring a distinctive repertoire of gene expression strategies. Functional mature messenger RNA is generated via the trans-splicing and polyadenylation processing of constitutively transcribed polycistronic units. Recently, trans-splicing of pre-small subunit ribosomal RNA in the 5' external transcribed spacer region and of precursor tRNAsec have been described. Here, we used a previously validated semi-nested reverse transcription-polymerase chain reaction strategy to investigate internal transcribed spacer (ITS) I acceptor sites in total RNA from Leishmania (Leishmania) amazonensis. Two distinct spliced leader-containing RNAs were detected indicating that trans-splicing reactions occur at two AG acceptor sites mapped in this ITS region. These data provide further evidence of the wide spectrum of RNA molecules that act as trans-splicing acceptors in trypanosomatids.

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

Correlation of meta 1 expression with culture stage, cell morphology and infectivity in Leishmania (Leishmania) amazonensis promastigotes

The parasitic protozoan Leishmania (Leishmania) amazonensis alternates between mammalian and insect hosts. In the insect host, the parasites proliferate as procyclic promastigotes andthen differentiate into metacyclic infective forms. The meta 1 gene is preferentially expressed during metacyclogenesis. Meta 1 expression profile determination along parasite growth curves revealed that the meta 1 mRNA level peaked at the early stationary phase then decreased to an intermediate level. No correlation was observed between meta 1 expression and infectivity. Conversely, infectivity correlated with the increase of apoptotic cells in the late stationary phase.

Etiology of cutaneous leishmaniasis and anthropophilic vectors in Juruti, Pará State, Brazil / Etiologia da leishmaniose cutânea e vetores antropofílicos em Juruti, Estado do Pará, Brasil

In a preliminary study in Juruti, a mining municipality in western Pará State, Brazil, 12 out of 21 patients suspected of presenting cutaneous leishmaniasis showed positive PCR (SSUrDNA and G6PD): Leishmania (Viannia) braziliensis (9/12; 75 percent) and L. (V.) sp. (3/12; 25 percent). Entomological studies in the same location revealed the presence of 12 different phlebotomine species (n =105). One of the most common species was Lutzomyia (Psychodopygus) complexa (17 percent) which is both highly anthropophilic and a known vector of L. (V.) braziliensis in other regions of Pará. These preliminary findings should serve to guide future epidemiological surveillance in Juruti.

Em um estudo preliminar em Juruti, um município minerário na região oeste do Estado do Pará, Brasil, 12 de 21 pacientes suspeitos de possuírem leishmaniose cutânea tiveram PCRs positivas (SSUrDNA e G6PD): Leishmania (Viannia) braziliensis (9/12; 75 por cento) e Leishmania (Viannia) sp. (3/12; 25 por cento). Estudos entomológicos na mesma localidade revelaram a presença de 12 diferentes espécies de flebotomíneos (n = 105). Uma das espécies mais comuns foi Lutzomyia (Psychodopygus) complexa (17 por cento) que é altamente antropofílica e reconhecida vetora de L. (V.) braziliensis em outras regiões do Estado do Pará. Esses resultados preliminares servem como orientação para futura vigilância epidemiológica em Juruti.

RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Leishmania (Sauroleishmania) tarentolae has biotechnological potential for use as live vaccine against visceral leishmaniasis and as a system for the over expression of eukaryotic proteins that possess accurate post-translational modifications. For both purposes, new systems for protein expression in this non-pathogenic protozoan are necessary. The ribosomal RNA promoter proved to be a stronger transcription driver since its use yielded increased levels of recombinant protein in organisms of both genera Trypanosoma or Leishmania. We have evaluated heterologous expression systems using vectors with two different polypyrimidine tracts in the splice acceptor site by measuring a reporter gene transcribed from L. tarentolae RNA polymerase I promoter. Our data indicate that the efficiency of chloramphenicol acetyl transferase expression changed drastically with homologous or heterologous sequences, depending on the polypyrimidine tract used in the construct and differences in size and/or distance from the AG dinucleotide. In relation to the promoter sequence the reporter expression was higher in heterologous lizard-infecting species than in the homologous L. tarentolae or in the mammalian-infecting L. (Leishmania) amazonensis.

Ocurrence of co-infection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi in a dog in the state of Mato Grosso do Sul, Brazil

A natural case of co-infection by Leishmania and Trypanosoma is reported in a dog (Canis familiaris) in south- western state of Mato Grosso do Sul, Brazil. Both amastigote and trypomastigote forms were observed after Giemsa staining of cytological preparations of the dog's bone marrow aspirate. No parasite was detected using medium culture inoculation of the sample. DNA obtained from the bone marrow aspirate sample and from the blood buffy coat was submitted to polymerase chain reaction (PCR) with a set of rDNA-based primers S4/S12. The nucleotide sequence of the PCR product was identical to that of Trypanosoma (Trypanozoon) evansi. The S4/S12 PCR was then used as template in a nested-PCR using a specific Leishmania set S17/S18 as primers, to explain the amastigote forms. The nucleotide sequence of the new PCR product was identical to that of Leishmania (Leishmania) chagasi. This case, as far as we know, is the first report of a dog co-infected with these parasites, suggesting that besides L. (L.) chagasi, the natural transmission of T. (T.) evansi occurs in the area under study.

Pre-mRNA trans-splicing: from kinetoplastids to mammals, an easy language for life diversity

Since the discovery that genes are split into intron and exons, the studies of the mechanisms involved in splicing pointed to presence of consensus signals in an attempt to generalize the process for all living cells. However, as discussed in the present review, splicing is a theme full of variations. The trans-splicing of pre-mRNAs, the joining of exons from distinct transcripts, is one of these variations with broad distribution in the phylogenetic tree. The biological meaning of this phenomenon is discussed encompassing reactions resembling a possible noise to mechanisms of gene expression regulation. All of them however, can contribute to the generation of life diversity.

Genetic characterization of lactose fermentation and antibiotic resistance in plasmid-harboring enteric bacteria isolated from amazon amphibians

Bacterias aeróbicas gram-negativas foram isoladas de fezes de 20 anfíbios. Os animais foram capturados na regiäo que seria inundada para a formaçäo da represa da Usina Hidrelétrica de Tucuruí-PA. Entre as 420 colônias bacterianas analisadas, 123 (29%) eram portadoras de plasmídios, sendo: 89% capazes de fermentar a lactose, 41% resistentes à ampicilina, 6,5% à estreptomicina e 6,5% à tetraciclina, em níveis * 10 ug/ml. O gênero predominante entre as 123 amostras foi o Citrobacter (67%), seguido de Enterobacter (16%), Klebsiella (6%), Providencia (3%), Proteus (2%), Aeromonas (2%) e Salmonella (1%). Como grande parte dos plasmídios detectados tinha pesos moleculares suficientes para conter genes capazes de promover sua auto-transferência, a correlaçäo entre a presença dos mesmos e algumas características observadas foi investigada por conjugaçäo. A presença destes genes foi também analisada por segregaçäo natural e induzida, e por hibridizaçäo DNA-DNA, através de sondas radioativas. Os resultados näo demonstraram a presença de plasmídios R, Hly, ou Lac nestas amostras, mas foram encontradas evidências de que os caracteres de fermentaçäo de lactose e produçäo de hemolisinas säo medidos por genes presentes nos cromossomos destas bactérias