Parmi les parasites, les échinocoques revêtent une importance majeure en santé publique de par leur distribution mondiale et les maladies potentiellement très graves, qu’ils occasionnent. En Europe, les espèces présentes sont E. multilocularis, qui a un cycle sylvatique et E. granulosus spp., dont les hôtes sont essentiellement domestiques.En France, peu de données étaient disponibles quant à la distribution de ces espèces parasitaires lors de la création du LNR Echinococccus spp. en 2006. Une forte réduction supposée des zones et du niveau d’enzootie pour E. granulosus laissait envisager un risque zoonotique moindre voire désormais absent. S’agissant d’E. multilocularis, l’expansion constatée en Europe était également suspectée en France. La modification des techniques de parasitologie classique ainsi que le développement d’outils moléculaires par le LNR ont contribué à l’actualisation et à une meilleure connaissance de la distribution des espèces parasitaires en France.Une large étude de surveillance menée chez le renard avec la méthode SSCT validée par le LNR a révélé une expansion de la zone d’enzootie d’E. multilocularis jusqu’en Ille-et-Vilaine. L’analyse par microsatellite EmsB a permis d’estimer à plusieurs décennies la dispersion du parasite vers l’ouest et le nord à partir du foyer historique de l’est. Cette expansion a été confirmée à l’ouest chez des rongeurs aquatiques et rétrospectivement au sud chez le renard.Des études de surveillance d’E. granulosus en abattoir ont confirmé sa présence dans le sud de la France et en Corse. Les caractérisations moléculaires ont permis d’identifier E. granulosus sensu stricto dans le sud et E. canadensis G6-7 en Corse. Un plan de surveillance national en abattoir a ensuite montré la présence d’E. granulosus s.s. à travers l’ensemble du territoire continental. Les niveaux de prévalence d’infestation par E. granulosus s.s. estimés chez les ovins à 15,3 et chez les bovins à 8,3 cas pour 1 million de têtes abattues sont très inférieurs à ceux décrits il y a vingt ans. Deux foyers majeurs constitués par les Alpes pour E. granulosus s.s. et la Corse pour E. canadensis G6-7 demeurent, alors que l’identification d’E. ortleppi confirme le maintien de cette espèce zoonotique pourtant désormais rare en Europe.Les niveaux de prévalence d’infestation par E. multilocularis chez le chien et le chat en France confirment leurs importances mineures dans le maintien du cycle parasitaire. Le rôle zoonotique du chat semble négligeable d’après les observations d’infestations naturelles, corroborées par les données obtenues lors d’infestations expérimentales. La vermifugation régulière et adaptée des chiens, en particulier des chiens de chasse, apparait nécessaire. Cette recommandation de vermifugation canine s’applique également pour E. canadensis G6-7 en Corse, où l’accès aux viscères des porcs et des sangliers implique un volet sylvatique dans le cycle.La reconnaissance de l’expertise du LNR a permis d’initier des collaborations internationales qui ont en retour enrichi la diversité des situations épidémiologiques étudiées. Ainsi, l’étude de la diversité génétique d’E. multilocularis (microsatellite EmsB) a contribué à une meilleure compréhension de la dynamique d’expansion du parasite en Europe (Pologne, Suède, Danemark). L’étude des foyers d’hyper enzootie d’E. granulosus (Moldavie, Maroc, Algérie) et la caractérisation des espèces présentes et de leurs niveaux d’infestation chez les hôtes intermédiaires a permis d’envisager des actions de lutte adaptées. Le LNR a participé à l’expérimentation de méthodes de lutte contre E. multilocularis, démontrant la complexité des conditions requises pour leur efficacité tant par la vermifugation des renards que par la régulation de leur population. Au bilan, les nouvelles données épidémiologiques obtenues au cours des dix années de travaux ont permis d’aboutir à une meilleure appréhension du risque zoonotique actuel lié aux échinocoques en France / Among parasites, Echinococcus species are of major public health importance due to their worldwide distribution and the potential severity of the diseases they cause. In Europe, the endemic species are E. multilocularis, which has a sylvatic lifestyle, and E. granulosus spp., which the hosts are mainly domestic species. When the Echinococcus spp. NRL was created in France in 2006, few data were available on the distribution of these parasitic species. The implementation of health measures made it possible to consider a marked reduction of the endemic level and geographical distribution of E. granulosus and consequently a reduced or inexistent zoonotic risk. With regard to E. multilocularis, the parasite spread already observed in Europe was also suspected in France. The development of classical parasitology and molecular techniques for diagnosis and epidemiology has helped improve understanding of the distribution of these parasitic species in France.A large-scale surveillance study in foxes, using the SSCT method validated by the NRL, led to the description of significant westward and northward expansion from E. multilocularis’s historical endemic focus, which was estimated, using a spatio-temporal scenario deduced by EmsB microsatellite analysis, to have begun several decades ago. Molecular analyses of various types of animal samples confirmed its westward expansion in aquatic rodents. Its extension southward was confirmed thanks to fecal samples of foxes.Surveillance studies of E. granulosus at the slaughterhouse confirmed its presence in southern France and in Corsica. The first molecular characterization of this parasite in France resulted in the identification of E. granulosus sensu stricto in southern France and E. canadensis G6-7 in Corsica. The presence of E. granulosus s.s. throughout continental France was then observed on the basis of a national surveillance study at the slaughterhouse. The prevalence level of infection by E. granulosus s.s. estimated per million was 15.3 cases in sheep and 8.3 cases in cattle which was much lower than that described 20 years ago. The two main endemic foci – the Alps for E. granulosus s.s. and Corsica for E. canadensis G6-7 – still exist, while the identification of E. ortleppi confirmed maintenance of this species despite its current rarity in Europe.In France, the low prevalence of E. multilocularis infection in dogs and cats was ascertained, confirming the minor contribution of these hosts to the lifecycle. The zoonotic role of the cat appears to be negligible based on observations of natural infection cases and on data obtained from experimental infection. The regular and adapted deworming of dogs, especially hunting dogs, appears to be necessary. This deworming recommendation is also relevant against E. canadensis G6-7 in Corsica where access to viscera of pigs and wild boar adds a sylvatic component at this lifecycle.An acknowledgment of the NRL’s expertise led to several international collaborations which in turn contributed to the diversity of the epidemiological situations studied. Thus, the study of the genetic diversity of E. multilocularis by EmsB microsatellite led to a better understanding of the parasite’s expansion dynamics throughout Europe. The study of foreign foci of E. granulosus led to characterization of the endemic parasitic species and of their prevalence levels in intermediate hosts, which made it possible to plan appropriate control measures. The NRL participated in the testing of control methods against E. multilocularis, including deworming of foxes and population regulation, which demonstrated the complexity of the conditions required for these methods to be effective.The epidemiological data obtained over ten years of studies has led to a better understanding of the current zoonotic risk associated with the Echinococcus species in France

La equinococosis quística representa una grave zoonosis parasitaria en nuestro país y otros países en desarrollo dedicados a la ganadería. El agente causante es el céstodo Echinococcus granulosus, cuyo estadio adulto se desarrolla en el intestino delgado de hospederos definitivos (caninos) y el desarrollo del estadio larval se presenta principalmente en el hígado y pulmón de hospederos intermediarios como el ganado ovino. Una vacuna que proteja a los perros permitiría disminuir la biomasa parasitaría y la carga de huevos que infecta al rebaño. Por ello se evaluó la imnunoprotección contra E. granulosus empleando de antígenos de superficie como proteínas de membrana y productos metabólicos de excreción-secreción de protoescólices, y parásitos adultos. Las proteínas de membrana fueron obtenidas por extracción con Tritón x-114 y los productos de excreción/secreción (E/S) obtenidos por cultivo in vitro (Medio HAM F12). Además, se obtuvo antígeno total de la tenia adulta por sonicación. Se usó Quil A (50 g/ml) como adyuvante. Los antígenos fueron administrados por vía intranasal y se emplearon 12 perros divididos en cuatro grupos de tratamiento: control, E/S, proteínas de membrana (PM) y proteína total. Los animales recibieron tres inmunizaciones en intervalos de 15 días; se enfrentaron con 150000 protoescólices viables por vía oral, 15 días después de la última inmunización. Los perros fueron sacrificados entre 49-53 días después del enfrentamiento. Se evaluó la carga parasitaria y la presencia de huevos. Los grupos E/S y PM tuvieron menor carga parasitaria y ausencia de huevos en comparación al control, aunque no hubo diferencia estadística significativa. Los resultados muestran la posibilidad de continuar estudios con estos antígenos, ya que la inhibición del desarrollo embrionario es crucial para detener la transmisión a hospederos intermediarios y al hombre. -- Palabras clave: Echinococcus granulosus, protoescólex, inmunización, antígeno excretorio-secretorio, equinococosis quística. / Cystic echinococcosis is a serious parasitic zoonosis in our country and other developing countries dedicated to livestock. The causative agent is the tapeworm Echinococcus granulosus, whose adult stage takes place in the small intestine of definitive hosts (dogs) and the development of the larval stage occurs mainly in the liver and lung of intermediate hosts such as sheep. A vaccine to protect dogs would reduce the parasitic biomass and burden of eggs that infecting the flock. Therefore imnunoprotección was evaluated against E.granulosus using surface antigens as membrane proteins and metabolic products of excretion-secretion protoscoleces and adult worms. Membrane proteins were obtained by extraction with Triton x-114 and the products of excretion/secretion (E/S) obtained by in vitro culture (Medium HAM F12). Furthermore, the total antigen obtained had grown by sonication. Quil A was used (50 g/ml) as an adjuvant. The antigens were administered intranasally and 12 dogs were used divided into four treatment groups: control, E/S, membrane protein (PM) and total protein. Animals received three immunizations at 15 days intervals; protoscoleces faced viable 150000 orally, 15 days after the last immunization. The dogs were sacrificed between 49-53 days after challenge. Parasite load was evaluated and the presence of eggs. Groups E/S and PM had lower parasite load and absence of eggs compared to the control, although there was no statistically significant difference. The results show the possibility of continuing studies with these antigens, since inhibition of embryo development is critical to stop the transmission intermediate hosts and man. Key Words: Echinococcus granulosus, protoscoleces, immunization, excretory-secretory antigen, cystic echinococcosis.

No description available.

El antígeno B (EgAgB) es una lipoproteína presente en la larva del parásito cestodo Echinococcus granulosus, agente causante de la zoonosis conocida como hidatidosis, enfermedad endémica en nuestra región. Esta proteína pertenece a una familia de proteínas exclusivas de cestodos que unen ligandos hidrofóbicos, conocidas como HLBPs. A nivel proteico, el EgAgB está formado por subunidades de ~8 kDa codificadas por genes polimórficos pertenecientes a 5 subfamilias distintas (EgAgB8/1 a EgAgB8/5); mientras que su componente lipídico está formado por una amplia variedad de lípidos, incluyendo lípidos neutros y polares. El hecho de que muchos de estos lípidos no pueden ser sintetizados de novo por el parásito hace suponer que el EgAgB podría estar involucrado en la adquisición de estos compuestos desde el hospedador, como ha sido propuesto para otras HLBPs. Esto implicaría que el EgAgB interactué con componentes del hospedador para adquirir estos ligandos. En este sentido, existe evidencia de que el EgAgB es capaz de interactuar con células del sistema inmune, modulando su respuesta, lo que a su vez favorecería el establecimiento y permanencia del parásito. Sin embargo, para poder comprender los mecanismos asociados a esta modulación a nivel molecular y determinar si la proteína es capaz de transportar e intercambiar los lípidos, es imprescindible avanzar en el conocimiento estructural y funcional de esta partícula compleja. Con este fin, durante el desarrollo de este trabajo se purificaron las subunidades recombinantes EgAgB8/1, EgAgB8/2 y EgAgB8/3, logrando obtenerlas de forma libre de lípidos. Se logró determinar que estas subunidades son capaces de interaccionar consigo mismas aún en ausencia de lípidos, formando oligómeros de entre 40 y 60 kDa, distintos a los que se han reportado previamente en la literatura en presencia de los ligandos. Por otro lado, se analizó la capacidad de distintos ligandos lipídicos de unirse a estas subunidades y se pudieron determinar constantes de disociación en el orden submicromolar empleando antroiloxi-derivados de ácidos grasos. Más aún, se evaluó la capacidad de las subunidades EgAgB8/2 y EgAgB8/3 de transferir estos derivados hacia membranas fosfolipídicas modelo. Utilizando distintos tipos de vesículas se pudo establecer que ambas subunidades son potencialmente capaces de transferir los ácidos grasos. En el caso de las subunidad EgAgB8/2, la transferencia ocurriría por un mecanismo que involucra un contacto directo con la membrana, mientras que en el caso de EgAgB8/3 la cinética de la transferencia estaría determinada por la disociación del complejo proteína-ligando. Asimismo, para ambas proteínas se pudo determinar que las interacciones electroestáticas tienen importancia en el mecanismo, dado que la transferencia se ve favorecida en el caso de vesículas con mayor carga neta negativa, como ocurre en el caso de vesículas ricas en cardiolipina. Por otro lado, con el fin de evaluar la capacidad de las subunidades libres de lípidos de interactuar con células del sistema inmune, particularmente con monocitos y macrófagos, se encontró que las subunidades EgAgB8/1 y EgAgB8/3 libres de lípidos son reconocidas por estas células, mientras que la subunidad EgAgB8/2 no presenta unión a los monocitos y es pobremente reconocida por los macrófagos. Asimismo, se pudo determinar que la fosfatidilcolina que estaría expuesta en la partícula nativa de EgAgB participaría en la unión, posiblemente modificando la forma en que se exponen las subunidades de EgAgB para que sean reconocidas por las células. En conjunto, los resultados obtenidos en este trabajo sugieren que la partícula de EgAgB puede ser reconocida por células del sistema inmune a través de algunas de sus subunidades y el hecho de que en proximidad de una membrana fosfolipídica algunas subunidades pueden ser capaces de transferir sus ligandos, apoya la idea de que EgAgB pueda intercambiar lípidos interaccionando con células del hospedador.

In this study different molecular techniques are contrasted (RAPD's, allozyme, sequencing mtDNA, sequencing ribosomal spacers) and appropriate analytical methods (allelic and infinite-sites approaches; inbreeding and coalescent models) used for estimating population genetic parameters in parasites. A range of population genetic questions at different scales were chosen to emphasise the importance of tailoring techniques and analytical methods to the particular question being investigated. The realisation that each question formulated has a particular scale means the appropriate technique and markers must be useful at that scale to attempt to answer the question. The useful scale of a technique depends several factors including the region of DNA examined, the density of sampling of the technique, and the mode of evolution of the markers. Each technique will produce a useful range of variability. Below the lower limit there is no variation, above the upper limit the variation is too high to produce useful comparisons. Parasites are of interest for many reasons, primarily because they can cause disease and thus impact on their host's population dynamics. They are often closely associated with their hosts and may undergo co-evolution, as well as causing an ongoing immunological "arms race" with their hosts. The parasitic mode of live is found throughout nearly all taxonomic groupings and thus classical models of population genetics based on sexual, diploid vertebrates do not fit well with the entire diversity of parasite groups. Genetic diversity within and among populations of Echinococcus granulosus was examined contrasting a RAPD dataset with an allozyme dataset. Two models of variation in Echinococcus have been proposed, those of Smyth and Rausch, and the expected genetic structure from each was compared to the observed genetic structure. The premise of Smyth’s model, predominant self-fertilisation, was supported, but the resultant pattern of genetic variation followed Rausch’s model. RAPD data, being dominant, present challenges to analysis. An approach to overcome this dominance problem and allow standard allelic frequency analysis is described using the selfing rate estimated from allozyme data. The RAPD data were also analysed using both band-sharing and nucleotide diversity approaches. A population genetic study of Ostertagia ostertagi in the USA was extended to two different scales: within an Australian state and between the USA and Australian continents. Three alternative explanations for the observed discrepancy between genetic structure and differentiation in an important biological trait, hypobiosis, were explored. A number of programs and analyses were compared including coalescent geneflow estimates. Variation among multiple copies of two spacer regions of rDNA was examined within individuals of Ostertagia ostertagi. Both the intergenic spacer and internal transcribed spacer 1 regions were found to include repeat regions, with different numbers of repeats creating length differences in clones from the same worm. Multi-copy genes present extra challenges in analysis to ensure that only homologous copies are being compared. Many studies fail to look for variation within populations or within individuals. The two major conclusions from these examples are that: 1). The study of variation necessarily involves an implicit scale, and markers must be chosen that are appropriate to the question being explored. 2). Using several methods of analysis of genetic data allows contrasts to be made, and if different methods produce similar results gives much more confidence in the conclusions drawn. Incongruence in results leads to new questions and reexamination of the assumptions of each analysis.

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-09-17T12:00:44Z No. of bitstreams: 1 TESE-ELSR.pdf: 8566121 bytes, checksum: 9b14e91fc47ab442c01443be29b2de9e (MD5) / Made available in DSpace on 2013-09-17T12:00:44Z (GMT). No. of bitstreams: 1 TESE-ELSR.pdf: 8566121 bytes, checksum: 9b14e91fc47ab442c01443be29b2de9e (MD5) Previous issue date: 2011-09-29 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Echinococcus granulosus, o menor cestóide, é distribuído por todo o mundo. Este parasito, sob a forma de larva, é responsável pela equinococose cística (EC) ou hidatidose, uma das zoonoses de maior importância médica e veterinária que ocasiona grandes perdas econômicas nas regiões pecuárias e agrícolas. E. granulosus é caracterizado por apresentar uma alta variabilidade intra-específica que é associada com hospedeiros intermediários diferentes. Dez variantes intra-específicas ou genótipos distintos (G1-G10) foram definidos com base na diversidade genética. A identificação de variantes ou genótipos circulantes em diferentes hospedeiros das regiões endêmicas da EC é epidemiologicamente importante, porque diferentes características biológicas entre as variantes individuais podem influenciar no padrão de ciclo de vida, especificidade do hospedeiro, o tempo de desenvolvimento, antigenicidade, dinâmica de transmissão, sensibilidade a agentes quimioterápicos e patogênese, com implicações importantes para a epidemiologia, diagnóstico, tratamento e controle de endemia. A região andina do Peru, que inclui as áreas de Puno, Junín, Arequipa, Cusco, Huanacavelica e Ayacucho, tem uma alta prevalência de EC. Para determinar as variantes ou genótipos de E. granulosus circulantes em hospedeiros intermediários das regiões endêmicas do Peru, foram coletadas amostras de cistos hidáticos de bovinos (44), ovinos (41) e humanos (14). Cistos hidáticos de alpaca (4) de Puno e porcos (8) de Ayacucho também foram incluídos neste estudo. DNA de todos os isolados (protoscólices e/ou camadas germinativas) de E. granulosus foram extraídos e usados para amplificar duas regiões de genes mitocondriais que codificam o citocromo C oxidase subunidade 1 (CO1) e NADH desidrogenase subunidade 1 (ND1). Produtos de PCR CO1 (450bp) e ND1 (550bp e 800bp) foram sequenciados e as sequências comparadas com as referências para E. granulosus encontrados na base de dados do GenBank. Análises filogenéticas também foram desenvolvidas. Sequências parciais do gene CO1 (375bp) e ND1 (483bp e 747bp) mostraram que todos os isolados de bovinos, ovinos, alpaca e 4/8 isolados de porcos correspondem ao genótipo G1 (linhagem da ovelha comum). Três isolados de ovelhas de Ayacucho mostraram polimorfismo de apenas um nucleotídeo indicando a presença de uma microvariante do genótipo G1 de E. granulosus, a G14. Quatro de oito sequências isoladas de porco de Ayacucho mostraram identidade com o genótipo G7 (linhagem suína) de E. granulosus. As análises filogenéticas das sequências dos genes CO1 e ND1 mostraram árvores semelhantes para ambos os genes, onde há um grupo albergando a maioria das sequências com o genótipo G1 e outro de quatro sequências isoladas de porco com o genótipo G7. Além disso, um grupo particular de três sequências isoladas de ovelhas provenientes de Ayacucho é agrupado com a microvariante G14. Os resultados obtidos neste estudo mostram a predominância do genótipo G1, em bovinos, ovinos, alpacas, porcos e seres humanos nas regiões endêmicas do Peru. A microvariante G14 foi identificada pela primeira vez na América do Sul. Dois genótipos G1 e G7 podem infectar porcos de Ayacucho. Os dados mostrados devem ser considerados para que as medidas de prevenção e controle da EC sejam verdadeiramente eficazes nessas regiões. / Echinococcus granulosus is a small cestode distributed worldwide and its larval stage is responsible for cystic echinococcosis (CE) or hydatidosis, a zoonosis of great medical and veterinary importance due to considerable economic losses of livestock in agricultural regions. E. granulosus is characterized by a high intraspecific variability that is associated with different intermediate hosts. Ten distinct genetic variants (G1-G10) have been defined based on molecular analysis. The identification of the genotypes circulating in different hosts from endemic regions of CE is epidemiologically important since each individual variant has different biological characteristics that can influence the life cycle pattern, host specificity, development rate, antigenicity, transmission dynamics, sensitivity to chemotherapeutic agents and pathology. These characteristics have important implications for the diagnosis, treatment and control of CE in endemic regions. The Andean region of Peru, which includes the areas of Puno, Junín, Arequipa, Cusco, Huanacavelica and Ayacucho, has a high prevalence of CE. To determine the genotypes of the E. granulosus circulating through intermediate hosts within these endemic regions of Peru, samples of hydatid cysts were collected from cattle (44), sheep (41) and humans (14). Hydatid cyst of alpaca (4) from Puno and pigs (8) from Ayacucho also were included in this study. DNA was extracted from the protoscolex and/or germinal layers in all of the isolates and used as a template to amplify regions of two mitochondrial genes that encode cytochrome C oxidase subunit 1 (CO1) and NADH dehydrogenase subunit 1 (ND1). PCR products of CO1 (450bp) and ND1 (550bp and 800bp) were sequenced and the sequences compared with the reference sequences of E. granulosus deposited in the GenBank data base. Phylogenetic analysis were also developed. Partial sequences of the CO1 gene (375bp) and ND1 gene (483 bp and 747 bp) showed that all isolates from cattle, sheep, alpaca and 4/8 isolated from pigs correspond to the G1 genotype (common sheep strain). Three isolates of sheep from Ayacucho showed a single nucleotide polymorphism indicating the presence of a microvariant of the G1 genotype of E. granulosus, the G14. Four of eight sequences of pig isolates from Ayacucho showed identity with the G7 genotype (pig strain) of E. granulosus. Phylogenetic analysis of the sequences of the genes CO1 and ND1 showed trees similar for both genes, where there is a grouping of most of the sequences with the G1 genotype and the other four sequences isolated from pigs with genotype G7. In addition, a particular group of sequences isolated from three sheep from Ayacucho is grouped with the G14 microvariante. The results obtained in this study show a predominance for the G1 genotype in cattle, sheep, alpacas, pigs and humans within endemic regions of Peru, the first identification of the microvariante G14 in South America and demonstrate that the genotypes G1 and G7 can infect pigs. The data shown should be considered for the development and implementation of effective programs in endemic regions of Peru for the control and prevention of EC.

Echinococcus granulosus é um parasito cestódeo e o agente etiológico da hidatidose cística. O desenvolvimento dos vermes adultos a partir dos protoscoleces é, provavelmente, desencadeada por diferentes estímulos que são impostos pelos hospedeiros, os quais provocam mudanças moleculares e morfológicas no parasito. No entanto, os mecanismos responsáveis por essas variações não estão totalmente esclarecidos. Para encontrar genes e proteínas possivelmente envolvidos com as alterações fenotípicas observadas durante o desenvolvimento estrobilar de E. granulosus, relatamos aqui o perfil transcritômico e a detecção de proteínas recémsintetizadas após a indução in vitro de protoescólices ao desenvolvimento estrobilar. Os protoescólices foram tratados com pepsina e mantidos em meio bifásico contendo taurocolato para induzir ao desenvolvimento estrobilar. Azidohomoalanina, aminoácido análogo de metionina, foi adicionado para a incorporação metabólica nas proteínas recém-sintetizadas, que foram visualizadas por microscopia confocal e identificadas por espectrometria de massas Paralelamente, o RNA total foi isolado utilizando o reagente TRIzol e as bibliotecas geradas foram sequenciadas na plataforma Illumina MiSeq. Nós identificamos 23 proteínas detectadas exclusivamente na presença de estímulos para o desenvolvimento estrobilar (SSD) e 28 na ausência desses estímulos (NSD). Também encontramos 75 proteínas diferencialmente expressas entre as duas condições (34 em SSD e 41 em NSD). Na análise dos dados de RNAseq, 818 genes foram identificados como diferencialmente expressos pelo software GFOLD. De forma geral, genes e proteínas com funções moleculares de transdução de sinal, metabolismo e modificações de proteínas foram observadas com a progressão do desenvolvimento estrobilar. Assim, este estudo fornece informações sobre genes e proteínas que podem ter um papel chave para o desenvolvimento do parasito, além de serem alvo de estudos futuros. / Echinococcus granulosus is a cestode parasite and the etiological agent of the cystic hydatid disease. The development of the adult worms from protoscoleces is probably triggered by different stimuli imposed by the hosts, which lead to molecular and morphological changes. However, the mechanisms underlying these variations remain largely unclear. In order to find genes and proteins possibly involved with the phenotypic changes during the strobilar development, we reported here the transcriptomic profile and the detection of newly synthesized proteins after protoscoleces in vitro induction to strobilar development. Protoscoleces were treated with pepsin and maintained in biphasic medium containing taurocholate to induce the strobilar development. The methionine analogue azidohomoalanine was added for metabolic incorporation in the newly synthesized proteins that were visualized by confocal microscopy and identified by mass spectrometry.In parallel, total RNA from parasite material was isolated using TRIzol reagent and the generated libraries were sequenced on Illumina MiSeq platform. We identified 23 proteins present only after stimuli for strobilar development (SSD) and 28 in absence of these stimuli (NSD). We also found 75 differentially expressed proteins (34 SSD and 41 NSD). In the RNA-seq analysis, 818 differentially expressed genes were identified by the GFOLD software. Gene transcripts and proteins with molecular functions of signal transduction, metabolism and protein modifications were observed with progression of development. This study provides insights on searching for the key genes and proteins that may be important for the correct parasite development and be target for further studies.

Echinococcus granulosus é um parasito cestódeo e o agente etiológico da hidatidose cística. O desenvolvimento dos vermes adultos a partir dos protoscoleces é, provavelmente, desencadeada por diferentes estímulos que são impostos pelos hospedeiros, os quais provocam mudanças moleculares e morfológicas no parasito. No entanto, os mecanismos responsáveis por essas variações não estão totalmente esclarecidos. Para encontrar genes e proteínas possivelmente envolvidos com as alterações fenotípicas observadas durante o desenvolvimento estrobilar de E. granulosus, relatamos aqui o perfil transcritômico e a detecção de proteínas recémsintetizadas após a indução in vitro de protoescólices ao desenvolvimento estrobilar. Os protoescólices foram tratados com pepsina e mantidos em meio bifásico contendo taurocolato para induzir ao desenvolvimento estrobilar. Azidohomoalanina, aminoácido análogo de metionina, foi adicionado para a incorporação metabólica nas proteínas recém-sintetizadas, que foram visualizadas por microscopia confocal e identificadas por espectrometria de massas Paralelamente, o RNA total foi isolado utilizando o reagente TRIzol e as bibliotecas geradas foram sequenciadas na plataforma Illumina MiSeq. Nós identificamos 23 proteínas detectadas exclusivamente na presença de estímulos para o desenvolvimento estrobilar (SSD) e 28 na ausência desses estímulos (NSD). Também encontramos 75 proteínas diferencialmente expressas entre as duas condições (34 em SSD e 41 em NSD). Na análise dos dados de RNAseq, 818 genes foram identificados como diferencialmente expressos pelo software GFOLD. De forma geral, genes e proteínas com funções moleculares de transdução de sinal, metabolismo e modificações de proteínas foram observadas com a progressão do desenvolvimento estrobilar. Assim, este estudo fornece informações sobre genes e proteínas que podem ter um papel chave para o desenvolvimento do parasito, além de serem alvo de estudos futuros. / Echinococcus granulosus is a cestode parasite and the etiological agent of the cystic hydatid disease. The development of the adult worms from protoscoleces is probably triggered by different stimuli imposed by the hosts, which lead to molecular and morphological changes. However, the mechanisms underlying these variations remain largely unclear. In order to find genes and proteins possibly involved with the phenotypic changes during the strobilar development, we reported here the transcriptomic profile and the detection of newly synthesized proteins after protoscoleces in vitro induction to strobilar development. Protoscoleces were treated with pepsin and maintained in biphasic medium containing taurocholate to induce the strobilar development. The methionine analogue azidohomoalanine was added for metabolic incorporation in the newly synthesized proteins that were visualized by confocal microscopy and identified by mass spectrometry.In parallel, total RNA from parasite material was isolated using TRIzol reagent and the generated libraries were sequenced on Illumina MiSeq platform. We identified 23 proteins present only after stimuli for strobilar development (SSD) and 28 in absence of these stimuli (NSD). We also found 75 differentially expressed proteins (34 SSD and 41 NSD). In the RNA-seq analysis, 818 differentially expressed genes were identified by the GFOLD software. Gene transcripts and proteins with molecular functions of signal transduction, metabolism and protein modifications were observed with progression of development. This study provides insights on searching for the key genes and proteins that may be important for the correct parasite development and be target for further studies.

Memoria para optar al Título Profesional de Médico Veterinario / La equinococosis, en una enfermedad zoonótica que se presenta en la mayoría de los continentes, desarrollándose especialmente en países con producción ganadera. Las características del ciclo vital de E. granulosus predispone a que el ser humano se vea afectado por éste desarrollando uno o varios quistes hidatídicos, pudiendo llevarlo incluso a la muerte. En la Región Metropolitana, la comuna de Melipilla se encuentra en segundo lugar en cuanto a la tasa de egresos hospitalarios de hidatidosis comprendida entre los años 2001 y 2008. El objetivo de este estudio fue determinar la prevalencia de equinococosis en perros pertenecientes a predios asesorados por el Programa de Desarrollo Local (PRODESAL) en el área rural del sector sur de la comuna de Melipilla, Región Metropolitana, Chile, en el año 2012. Se utilizaron muestras de heces frescas de perros pertenecientes a 64 usuarios con producción ganadera asesorados por PRODESAL distribuidos en 7 localidades de la comuna de Melipilla. Las muestras fueron procesadas por la técnica de reacción de polimerasa en cadena (PCR) en el laboratorio clínico “CAMPVS” ubicado en Santiago. De 234 perros muestreados, 17 de ellos (7,2%) resultaron positivos a la presencia de E. granulosus, por lo contrario en 217 perros (92,8%) no se identificó la presencia de dicho parásito. Además, de un total de 7 localidades muestreadas, en 4 de ellas se identificó presencia de E. granulosus en perros. También, se comprobó que no existe relación entre la presencia del parásito y el sexo (p>0,05) ni edad (p>0,05). Es importante contar con esta información para poder establecer programas de prevención y control de esta parasitosis en la población canina de la comuna de Melipilla y así, disminuir el número de animales y personas afectadas por ella

Echinococcus granulosus é um parasito cestódeo e o agente etiológico da hidatidose cística. O desenvolvimento dos vermes adultos a partir dos protoscoleces é, provavelmente, desencadeada por diferentes estímulos que são impostos pelos hospedeiros, os quais provocam mudanças moleculares e morfológicas no parasito. No entanto, os mecanismos responsáveis por essas variações não estão totalmente esclarecidos. Para encontrar genes e proteínas possivelmente envolvidos com as alterações fenotípicas observadas durante o desenvolvimento estrobilar de E. granulosus, relatamos aqui o perfil transcritômico e a detecção de proteínas recémsintetizadas após a indução in vitro de protoescólices ao desenvolvimento estrobilar. Os protoescólices foram tratados com pepsina e mantidos em meio bifásico contendo taurocolato para induzir ao desenvolvimento estrobilar. Azidohomoalanina, aminoácido análogo de metionina, foi adicionado para a incorporação metabólica nas proteínas recém-sintetizadas, que foram visualizadas por microscopia confocal e identificadas por espectrometria de massas Paralelamente, o RNA total foi isolado utilizando o reagente TRIzol e as bibliotecas geradas foram sequenciadas na plataforma Illumina MiSeq. Nós identificamos 23 proteínas detectadas exclusivamente na presença de estímulos para o desenvolvimento estrobilar (SSD) e 28 na ausência desses estímulos (NSD). Também encontramos 75 proteínas diferencialmente expressas entre as duas condições (34 em SSD e 41 em NSD). Na análise dos dados de RNAseq, 818 genes foram identificados como diferencialmente expressos pelo software GFOLD. De forma geral, genes e proteínas com funções moleculares de transdução de sinal, metabolismo e modificações de proteínas foram observadas com a progressão do desenvolvimento estrobilar. Assim, este estudo fornece informações sobre genes e proteínas que podem ter um papel chave para o desenvolvimento do parasito, além de serem alvo de estudos futuros. / Echinococcus granulosus is a cestode parasite and the etiological agent of the cystic hydatid disease. The development of the adult worms from protoscoleces is probably triggered by different stimuli imposed by the hosts, which lead to molecular and morphological changes. However, the mechanisms underlying these variations remain largely unclear. In order to find genes and proteins possibly involved with the phenotypic changes during the strobilar development, we reported here the transcriptomic profile and the detection of newly synthesized proteins after protoscoleces in vitro induction to strobilar development. Protoscoleces were treated with pepsin and maintained in biphasic medium containing taurocholate to induce the strobilar development. The methionine analogue azidohomoalanine was added for metabolic incorporation in the newly synthesized proteins that were visualized by confocal microscopy and identified by mass spectrometry.In parallel, total RNA from parasite material was isolated using TRIzol reagent and the generated libraries were sequenced on Illumina MiSeq platform. We identified 23 proteins present only after stimuli for strobilar development (SSD) and 28 in absence of these stimuli (NSD). We also found 75 differentially expressed proteins (34 SSD and 41 NSD). In the RNA-seq analysis, 818 differentially expressed genes were identified by the GFOLD software. Gene transcripts and proteins with molecular functions of signal transduction, metabolism and protein modifications were observed with progression of development. This study provides insights on searching for the key genes and proteins that may be important for the correct parasite development and be target for further studies.