Morphological effects on helminth parasites caused by herbicide under experimental conditions / Efeitos morfológicos em helminto parasito causado por herbicida em condições experimentais

Abstract Helminth parasites have been studied as potential accumulators for different pollutants. Echinostoma paraensei is a foodborne trematode whose vertebrate host, the rodent Nectomys squamipes, is naturally exposed to environmental pesticides. However, little information exists regarding the pesticide's effects on helminths. This study investigated the morphological effects on the trematode, E. paraensei, after experimental Roundup® herbicide exposure, in concentrations below those recommended for agricultural use. After two hours of exposure, scanning electron microscopy (SEM) showed changes to the tegument, such as furrowing, shrinkage, peeling, spines loss on the peristomic collar, and histopathological evidence of altered cells in the cecum and acinus vitelline glands with vacuoles and structural changes to the muscular layers. Glycidic content was decreased, primarily in the connective tissue. As E. paraensei is an intestinal parasite of the semi-aquatic wild rodent, N. squamipes, it is predisposed to pesticide exposure resulting from agricultural practices. Therefore, we emphasize the need to evaluate its impact on helminth parasites, due to their pivotal role in regulating host populations.

Resumo Helmintos parasitos tem sido estudados como acumuladores potenciais para diferentes poluentes. O trematódeo E. paraensei tem como hospedeiro vertebrado o roedor Nectomys squamipes naturalmente exposto a pesticidas no meio ambiente. No entanto, pouca informação está disponível sobre os efeitos dos pesticidas em helmintos parasitos. O presente estudo investigou, em condições experimentais, os efeitos morfológicos no trematódeo E. paraensei após a exposição ao herbicida Roundup®, em concentrações abaixo das recomendadas para a utilização agrícola. A microscopia eletrônica de varredura (MEV) mostrou após duas horas de exposição, alterações no tegumento, como enrugamento, contração e descamação com perda de espinhos no colar peristômico e análise histopatológica evidenciou células do ceco alteradas, as glândulas vitelínicas com vacúolos e mudanças estruturais nas camadas musculares. Diminuição do conteúdo glicídico, principalmente no tecido conjuntivo, também foi observado. Considerando a predisposição à exposição a pesticidas agrícolas de N. squamipes infectado por E. paraensei, são necessários estudos para avaliar o impacto de tais resíduos frente aos helmintos e seus hospedeiros.

Protection mediated by chemokine CXCL10 in BALB/c mice infected by Leishmania infantum

BACKGROUND Visceral leishmaniasis (VL) caused by Leishmania infantum is characterised by the loss of the ability of the host to generate an effective immune response. Chemokines have a direct involvement in the pathogenesis of leishmaniasis, causing a rapid change in the expression of these molecules during infection by Leishmania. OBJECTIVES Herein, it was investigated the role of CXCL10 in controlling infection by L. infantum. METHODS RAW 264.7 macrophages were infected with L. infantum in vitro and treated or not with CXCL10 (25, 50 and 100 ng/mL). Parasite load, as well as nitric oxide (NO), IL-4 and IL-10 production were assessed at 24 and 48 h after infection. In vivo, BALB/c mice were infected and treated or not with CXCL10 (5 μg/kg) at one, three and seven days of infection. Parasite load, IFN-g, IL-4, TGF-β and IL-10 were evaluated one, seven and 23 days post treatment. FINDINGS In vitro, CXCL10 reduced parasitic load, not dependent on NO, and inhibited IL-10 and IL-4 secretion. In vivo, CXCL10 was able to reduce the parasite load in both liver and spleen, four weeks after infection, representing a higher decrease in the number of parasites in these organs, also induced IFN-γ at day 23 after treatment, correlating with the decrease in parasite load, and reduced IL-10 and TGF-β. MAIN CONCLUSIONS This study suggests a partial protective role of CXCL10 against L. infantum, mediated by IFN-g, not dependent on NO, and with suppression of IL-10 and TGF-β. These data may provide information for the development of new approaches for future therapeutic interventions for VL.

Biofilm production and beta-lactamic resistance in Brazilian Staphylococcus aureus isolates from bovine mastitis

Abstract Staphylococcus spp. play an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant species due to the production of virulence factors such as slime, which is required for biofilm formation. This study aimed to evaluate biofilm production and its possible relation to beta-lactamic resistance in 20 S. aureus isolates from bovine mastitic milk. The isolates were characterized by pheno-genotypic and MALDI TOF-MS assays and tested for genes such as icaA, icaD, bap, agr RNAIII, agr I, agr II, agr III, and agr IV, which are related to slime production and its regulation. Biofilm production in microplates was evaluated considering the intervals determined along the bacterial growth curve. In addition, to determine the most suitable time interval for biofilm analysis, scanning electron microscopy was performed. Furthermore, genes such as mecA and blaZ that are related to beta-lactamic resistance and oxacillin susceptibility were tested. All the studied isolates were biofilm producers and mostly presented icaA and icaD. The Agr type II genes were significantly prevalent. According to the SEM, gradual changes in the bacterial arrangement were observed during biofilm formation along the growth curve phases, and the peak was reached at the stationary phase. In this study, the penicillin resistance was related to the production of beta-lactamase, and the high minimal bactericidal concentration for cefoxitin was possibly associated with biofilm protection. Therefore, further studies are warranted to better understand biofilm formation, possibly contributing to our knowledge about bacterial resistance in vivo.

O ensino de pós-graduação no IOC: segunda fase - 1976 a 2011 / The post graduation teaching in IOC: second phase - 1976 to 2011

A segunda fase do ensino no Instituto Oswaldo Cruz coincide com o turbulento período que se seguiu ao "Massacre de Manguinhos". A criação da Fundação Instituto Oswaldo Cruz mudou profundamente o regime administrativo do IOC, fusionando a outras três instituições (ENSP, IFF e INERU). O ensino no Instituto manteve a filosofia de Oswaldo Cruz de integrar pesquisa e ensino para enfrentar os principais problemas de saúde pública. Neste sentido, os programas de pós-graduação expandiram-se e consolidaram-se com lugar de destaque no sistema de ensino superior do país.

O ensino no IOC: primeira fase - 1900 a 1969 / Teaching in IOC: first phase - 1900 to 1969

A vocação para o ensino e a formação de recursos humanos para a ciência e a saúde está presente desde a criação do Instituto Soroterápico Federal de Manguinhos, em 25 de maio de 1900. Com o objetivo inicial de produzir soros e vacinas para combater a peste bubônica na virada do século, o Instituto foi a primeira iniciativa científico-tecnológica do país, e também deu início a uma longa tradição de formação de pesquisadores e profissionais para a Saúde e a Ciência, antes da criação da pós-graduação no Brasil, tal como se conhece hoje. Por isso se fala de tríade estrutural da Fundação Oswaldo Cruz: dessas três vertentes, e assim são o desenvolvimento tecnológico, a informação e a comunicação em ciência e saúde, a assistência e os serviços de laboratórios de referência, a cooperação nacional e internacional. Referência em qualquer tema desde que a pesquisa realizada seja de qualidade e que nela se formem pessoas, com o sentido de retorno desses conhecimentos para a sociedade.

O Instituto Oswaldo Cruz: pesquisa, ensino e serviços para a saúde / The Instituto Oswaldo Cruz: research, teaching and services for health

Pesquisa e ensino associam-se para marcar a importância histórica do IOC para o país e para a Fiocruz, pontuando suas conquistas recentes, focando na prospecção científica para acompanhamento da geração de conhecimentos em respostas às necessidades do Brasil. Identificamos o aumento da qualidade da produção científica do IOC, a ampliação e o fortalecimento dos cursos de mestrado e doutorado, a realização de Fóruns Colegiados entre discentes e docentes, e o fortalecimento da Gestão Participativa para o debate das ações do Instituto. A vitalidade da cooperação internacional do IOC é outro aspecto a ser registrado, contribuindo para a formação de cientistas no continente africano e na América Latina, onde a política de solidariedade não reconhece fronteiras. Neste capítulo, faremos um passeio rápido por essa história, unindo seus dois pontos, o inicial e o atual, com contribuição ao entendimento do que estamos fazendo, por que, como e para onde queremos ir em Pesquisa e Ensino e em tudo que deriva deste duro e agradável trabalho.

Por que uma escola para a ciência e a saúde: 111 anos de ensino no IOC / Why a school for science and health: 111 years of education in IOC

Breve apresentação do Instituto Oswaldo Cruz e da história contada no livro.

Scanning electron microscopy on Gotocotyla acanthura (Monogenea, Gotocotylidae) from Pomatomus saltatrix (Ostheichthyes, Pomatomidae) in Brazil / Microscopia eletrônica de varredura de Gotocotyla acanthura (Monogenea, Gotocotylidae) parasita de Pomatomus saltatrix (Ostheichthyes, Pomatomidae) no Brasil

Gotocotyla acanthura (Parona & Perugia, 1896) Meserve, 1938 collected from the gills of Pomatomus saltatrix from the coast of the state of Rio de Janeiro state was analyzed using scanning electron microscopy (SEM). The study demonstrated the presence of a buccal cavity, a genital atrium on the ventral surface and a muscular structure on the dorsal surface at the level of the body constriction. An elongated haptor with 80 to 120 pedunculated clamps symmetrically distributed in two rows, with rib-like thickenings and a curved lappet bearing a pair of hooks at the posterior extremity of the body were also observed. The cirrus could be seen protruding from the genital atrium, armed with pectinate spines along its length and presenting up to eight pointed spines around the genital atrium.

Gotocotyla acanthura (Parona & Perugia, 1896) Meserve, 1938, coletado das brânquias de Pomatomus saltatrix do litoral do Estado do Rio de Janeiro, foi estudado por microscopia eletrônica de varredura (MEV). O estudo demonstrou a presença de uma cavidade bucal, átrio genital na superfície ventral e uma estrutura muscular na superficie dorsal ao nível da constricção do corpo. Na extremidade posterior do corpo, foi observado o haptor alongado, apresentando 80 a 120 pinças pedunculadas, distribuídas em duas fileiras simétricas e uma porção terminal curvada provida de um par de ganchos. O cirro foi visualizado projetando-se do átrio genital, armado com espinhos pectinados ao longo do seu comprimento, apresentando até 8 espinhos pontiagudos ao redor do átrio.

Detecção de fibronectina e laminina em cistos teciduais de toxoplasma gondii: ensaios imunocitoquímicos / Detection of fibronectin and laminin in toxoplasma gondii tissue cysts: immunocytochemical assays

Aims: To analyze the existence and distribution of some matrix proteins in tissue cysts of Toxoplasma gondii. Methods: Laminin and fibronectin in tissue cysts of Toxoplasma gondii were detected by confocal microscopy and transmission electron microscopy. Results: Ultrastructural immunocytochemistry showed both glycoproteins in the granular region of tissue cysts, cystic matrix, micronemes, rhoptries, dense granules and rarely at the membrane of bradyzoites of Toxoplasma gondii. Conclusions: The presence of both laminin and fibronectin in secretory organelles and in the apical region of bradyzoites suggests that exocytosis of these glycoproteins can contribute to their interaction with host cells, besides composing the cyst matrix of Toxoplasma gondii.

Análise da viabilidade e infectividade de taquizoítas de toxoplasma gondii em condições axênicas / Viability and infectivity analysis of toxoplasma gondii underaxenic conditions

Aims: To investigate the infectivity and viability of the tachyzoite form of Toxoplasma gondii maintained in axenic medium. Methods: Tachyzoites of Toxoplasma gondii isolated from infected mice were suspended in phosphate-buffered saline pH 7.2 or phosphate-buffered saline pH 7.2 plus 10% or 20% foetal calf serum, and incubated for 24 and for 48 hours at 37ºC. Afterwards the parasites were: (i) incubated with propidium iodide and analysed by flow cytometry, using the fluorescence-activated cell sorting (FACS) system; (ii) injected in mice to check the parasite viability in axenic conditions and, (iii) added to mouse embryonic fibroblasts to investigate their infectivity and ability to intracellular development. Results: Analysis by flow cytometry showed that Toxoplasma gondii tachyzoites maintained in phosphate-buffered saline supplemented with 20% foetal calf serum displayed high cellular viability. The parasites kept their infectivity in both the in vivo and the in vitro systems, respectively demonstrated by their ability to replicate in mice and to form rosettes in mouse embryonic fibroblasts. Conclusions: Our data open new perspectives for the study of different aspects of Toxoplasma gondii cell biology, including nutrition mechanisms, in vitro drug trials, or cellular and molecular studies to be performed directly on the parasite.

Objetivos: investigar a infectividade e a viabilidade de formas taquizoítas de Toxoplasma gondii mantidas em meio axênico. Métodos: taquizoítos de Toxoplasma gondii foram isolados de camundongos infectados, ressuspensos em tampão fosfato-salino pH 7,2 ou tampão fosfato-salino pH 7,2 mais soro fetal bovino a 10% ou 20% e incubados por 24 e 48 horas a 37ºC em atmosfera contendo 5% de CO2. A seguir os parasitos foram: (i) incubados com iodeto de propídio e analisados por citometria de fluxo utilizando a técnica de separação de células ativada por fluorescência (fluorescence activated cell sorting ? FACS); (ii) injetados em camundongos para analisar a infectividade dos parasitos mantidos em condições axênicas; e (iii) adicionados à cultura de fibroblastos de embrião de camundongo para investigar a sua capacidade infectiva e multiplicativa com formação de rosetas e lise celular. Resultados: a análise por citometria de fluxo mostrou que os taquizoítos de Toxoplasma gondii mantidos em tampão fosfato-salino suplementado com 20% de soro fetal bovino apresentaram elevada viabilidade celular. Os parasitos mantiveram sua infectividade nos sistemas in vivo e in vitro, respectivamente demonstrada por sua capacidade de se replicar em camundongos e de infectar culturas de fibroblastos. Conclusões: nossos dados abrem novas perspectivas para o estudo de diferentes aspectos da biologia do Toxoplasma gondii, incluindo mecanismos de nutrição, ensaios experimentais de drogas in vitro, ou estudos da biologia celular e molecular diretamente sobre o parasito.

Interaction and cystogenesis of Toxoplasma gondii within skeletal muscle cells in vitro

Infection by the protozoan parasite Toxoplasma gondii is widely prevalent in humans and animals. To prevent human infection, all meat should be well cooked before consumption, since the parasite is present in skeletal muscle. In this context, the use of skeletal muscle cells (SkMCs) as a cellular model opens up new approaches to investigate T. gondii-host cell interactions. Immunofluorescent detection of proteins that are stage-specific for bradyzoites indicated that complete cystogenesis of T. gondii in in vitro cultures of SkMCs occurs after 96 h of infection. Ultrastructural analysis showed that, after 48 h of interaction, there were alterations on the parasitophorous vacuole membrane, including greater thickness and increased electron density at the inner face of the membrane. The present study demonstrates the potential use of primary cultures of SkMCs to evaluate different molecular aspects of T. gondii invasion and cystogenesis and presents a promising in vitro model for the screening of drug activities toward tissue cysts and bradyzoites.

Spontaneous stage differentiation of mouse-virulent Toxoplasma gondii RH parasites in skeletal muscle cells: an ultrastructural evaluation

Although the predilection for Toxoplasma gondii to form cysts in the nervous system and skeletal and heart muscles has been described for more than fifty years, skeletal muscle cells (SkMCs) have not been explored as a host cell type to study the Toxoplasma-host cell interaction and investigate the intracellular development of the parasite. Morphological aspects of the initial events in the Toxoplasma-SkMC interaction were analysed and suggest that there are different processes of protozoan adhesion and invasion and of the subsequent fate of the parasite inside the parasitophorous vacuole (PV). Using scanning electron microscopy,Toxoplasma tachyzoites from the mouse-virulent RH strain were found to be attached to SkMCs by the anterior or posterior region of the body, with or without expansion of the SkMC membrane. This suggests that different types of parasite internalization occurred. Asynchronous multiplication and differentiation of T. gondii were observed. Importantly, intracellular parasites were seen to display high amounts of amylopectin granules in their cytoplasm, indicating that tachyzoites of the RH strain were able to differentiate spontaneously into bradyzoites in SkMCs. This stage conversion occurred in approximately 3 percent of the PVs. This is particularly intriguing as tachyzoites of virulent Toxoplasma strains are not thought to be prone to cyst formation. We discuss whether biological differences in host cells are crucial to Toxoplasma stage conversion and suggest that important questions concerning the host cell type and its relevance in Toxoplasma differentiation are still unanswered.

The life history of Pygidiopsis macrostomum Travassos, 1928 (Digenea: Heterophyidae)

The life history of the trematode Pygidiopsis macrostomum Travassos, 1928 is described for the first time. Rediae and cercariae were obtained from naturally infected snails Heleobia australis (d´Orbigny), a new first intermediate host. Metacercariae were found encysted in the mesenteries of three naturally infected guppies, Phalloptychus januarius (Hensel), Jenynsia multidentata (Jenyns) (new host records) and Poecilia vivipara Bloch and Schneider. Experimental infections were successfully completed in the intermediate hosts H. australis and Poe. vivipara reared in the laboratory and hamsters Mesocricetus auratus Waterhouse were utilised as a definitive host.

The effect of Bulgarian propolis against Trypanosoma cruzi and during its interaction with host cells

Propolis has shown activity against pathogenic microorganisms that cause diseases in humans and animals. The ethanol (Et-Blg) and acetone (Ket-Blg) extracts from a Bulgarian propolis, with known chemical compositions, presented similar activity against tissue culture-derived amastigotes. The treatment of Trypanosoma cruzi-infected skeletal muscle cells with Et-Blg led to a decrease of infection and of the intracellular proliferation of amastigotes, while damage to the host cell was observed only at concentration 12.5 times higher than those affecting the parasite. Ultrastructural analysis of the effect of both extracts in epimastigotes revealed that the main targets were the mitochondrion and reservosomes. Et-Blg also affected the mitochondrion-kinetoplast complex in trypomastigotes, offering a potential target for chemotherapeutic agents.

Morfologia comparada dos ovos e ninfas de Triatoma vandae Carcavallo, Jurberg, Rocha, Galvão, Noireau & Lent, 2002 e Triatoma williami Galvão, Souza & Lima, 1965 (Hemiptera, Reduviidae, Triatominae) / Comparative morphology of eggs and nymphs of Triatoma vandae Carcavallo, Jurberg, Rocha, Galvão, Noireau & Lent, 2002 and Triatoma williami Galvão, Souza & Lima, 1965

Eggs and all nymphs of these species were studied employing light microscopy (LM) and scanning electron microscopy (SEM). The major differences observed by LM in the eggs were related to the presence and the distribution of pores on the surface of their chorion. Morphological differences among three nymphal stages (1st, 3rd, and 5th) development of each species were observed. The differential characteristics are chromatic and in the shape of connexival spots. The ultrastructure of the ventral region of the head and the IX, X, and XI abdominal segments (anal tube) of the both species were described demonstrating morphological differences that can be used for diagnosis of the species.

An alternative technique to reveal polysaccharides in Toxoplasma gondii tissue cysts

Ultrathin sections of tissue cysts isolated from the brain of Toxoplasma gondii infected mice were submitted to two different methodologies derived from the periodic acid - Schiff's reagent (PAS) technique. The use of osmium tetroxide vapor as a developing agent of the aldehyde oxidation to reveal polysaccharides with periodic acid resulted in positive reaction in amylopectin granules in bradyzoites, as well as in the wall and matrix of the cysts, with excellent increment of the ultrastructural morphology. This technique can be used for study of T. gondii-host cell intracellular cycle, the differentiation tachyzoite-bradyzoite, and also for the formation of cysts into the host cells.

Comparative study of the stridulatory sulcus, buccula and rostrum of the nymphs of Triatoma guazu Lent & Wygodzinsky, 1979 and Triatoma jurbergi Carcavallo, Galvão & Lent, 1998 by scanning electron microscopy (Hemiptera, Reduviidae)

The ultrastructural morphology of the ventral region of the head (rostrum and buccula) and proesternum (stridulatory sulcus) of nymphs from the 1st to 5th instars of Triatoma guazu Lent & Wygodzinsky, 1979 and Triatoma jurbergi Carcavallo, Galvão & Lent, 1998 was described. Morphological differences between the two species and of the five nymphal stages development of each species were observed. These structures showed systematic differential characteristics of the studied species and may be used to increase their taxonomic range

Anionic sites, fucose residues and Class I human leukocyte antigen fate during interaction of Toxoplasma gondii with endothelial cells

Toxoplasma gondii invades and proliferates in human umbilical vein endothelial cells where it resides in a parasitophorous vacuole. In order to analyze which components of the endothelial cell plasma membrane are internalized and become part of the parasitophorous vacuole membrane, the culture of endothelial cells was labeled with cationized ferritin or UEA I lectin or anti Class I human leukocytte antigen (HLA) before or after infection with T. gondii. The results showed no cationized ferritin and UEA I lectin in any parasitophorous vacuole membrane, however, the Class I HLA molecule labeling was observed in some endocytic vacuoles containing parasite until 1 h of interaction with T. gondii. After 24 h parasite-host cell interaction, the labeling was absent on the vacuolar membrane, but presents only in small vesicles near parasitophorous vacuole. These results suggest the anionic site and fucose residues are excluded at the time of parasitophorous vacuole formation while Class I HLA molecules are present only on a minority of Toxoplasma-containig vacuoles

Descriçäo dos ovos e dos estádios ninfais de Triatoma jurbergi Carcavallo, Galväo & Lent, 1998 vistos através de microscopia optica e eletrônica de varredura (Hemiptera, Reduviidae) / Description of eggs and nymphs of Triatoma jurbergi Carcavallo, Galväo & Lent, 1998 through optical and scanning electron microscopy

Eggs and nymphs of Triatoma jurbergi were described using optical microscopy and scanning electron microscopy. T. jurbergi is a wild species, found in State of Mato Grosso (15ºS and 300 m.a.s.l), Brazil. Eggs showed the operculum and surface with pentagonal and hexagonal cells, with small fractures and punctuations randomly distributed. Differences were found in the five nymphal stages of T. jurbergi, that allow their to be distinguished from the similar species T. guazu. The diagnostic characters most useful for differentiation were the general color of the insect, abdomen shape and its length