Giardia lamblia é um protozoário que acomete mais comumente animais jovens e que convivem em grupos. Apesar da alta prevalência, nem todos animais apresentam a doença clínica. Mesmo assim, a giardíase tem importância epidemiológica por possuir um elevado potencial zoonótico. O presente estudo teve como objetivo determinar a freqüência de Giardia lamblia em cães no município de Canoas, RS, Brasil, através do Método de Faust e cols. (1939) e da Técnica de Coloração da Auramina. Os grupos experimentais foram divididos de acordo com a procedência e o sexo. Das 332 amostras analisadas com o Método de Faust e cols, a estimativa em ponto da freqüência obtida foi de 34,04%, podendo variar de 28,95 a 39,13%, dentro de um intervalo de confiança de 95%. Destas amostras, 40,96% foram positivas em animais de canil e 27,11% de rua. O Teste Exato de Fisher aplicado a esses dados revelou existir uma diferença significativa (p = 0,0107) entre as variáveis resultado e procedência. A variável sexo, neste método não apresentou diferença significativa em relação ao resultado (p = 0,8162) totalizando 33,11% de machos positivos e 34,08% de fêmeas infectadas com o parasita. Das 147 amostras realizadas com a Técnica de Coloração da Auramina, 23 foram positivas, totalizando 15,65%. A análise estatística através do Teste McNemar revelou existir diferença significativa entre as duas técnicas (p = 0,0004). O valor Kappa foi igual a 0,07, considerado como um grau de concordância fraco. Os resultados encontrados neste estudo nos permitem afirmar que o Método de Faust e cols. foi o mais adequado para o diagnóstico na infecção por Giardia lamblia, entre os métodos analisados.

Giardia lamblia, a worldwide cause of diarrhoea, must differentiate into environmentally resistant cysts for dissemination and completion of its life cycle. Although G. lamblia is an early diverging eukaryote, encystation involves many complex cellular changes including formation of the cyst wall that contains at least two cyst wall proteins, cyst wall proteins 1 and 2. Cwp genes are transcribed only during encystation. In this study, we examine the regulatory elements for the encystation-specific gene cwp2. The 64 bp immediately upstream of the cwp2 open reading frame (-64 to -1 relative to ATG) was shown to be sufficient for the encystation-specific expression of luciferase. To determine which region(s) within this 64 bp contributed to encystation-specific expression in vivo, a series of deletions were cloned into a Giardia luciferase expression vector and their ability to control encystation-specific expression of luciferase was assessed. Deletion of elements in the -64 to -23 region of the cwp2 promoter significantly increased expression of luciferase in vegetative trophozoites, suggesting that this area contains a negative cis-acting element. Deletions of elements from -23 to -10 led to decreased expression in encysting cells, suggesting that this region may contain positive cis-acting elements. When the A/T-rich initiator was deleted but the cis-acting elements (-64 to -10) were retained, encystation-specific expression of luciferase was maintained but an aberrant transcriptional start site was utilised. These results indicate that Giardia has developed a classic repressor mechanism(s) that allows tight, encystation-specific control by the cwp2 promoter.

Zur Klärung der Frage, welche Rolle die Annexinen-homologen alpha-Giardine 14 und 19 tatsächlich im Lebenszyklus des humanpathogenen Darmparasits Giardia lamblia einnehmen, wurden verschiedene molekularbiologische und biochemische Untersuchungen durchgeführt. Die Untersuchungen im Falle des alpha14-Giardins ergaben, dass das Protein nur in gewissen Regionen der membranumgebenen Bereiche der Flagellen als Perlenschnur-ähnliche Verdickungen vorlag. Zusätzlich konnte sowohl eine Assoziation mit den Mikrotubuli des Axonems als auch mit der Plasmamembran nachgewiesen werden. Bei der Fahndung nach direkten Interaktionspartnern des alpha14 belegten verschiedene Methoden eine Wechselwirkung zwischen der Ankyrin-Domäne einer Ser/Thr-Kinase und dem alpha14. Auch konnte eine Phosphorylierung des Giardins eindeutig bestätigt werden. Weitere Eigenschaften des alpha14 waren die Fähigkeit der Oligomerisierung als auch die Bindung an Glykosaminoglykane. Infolgedessen könnte das alpha14-Giardin eine durch Phosphorylierung regulierte MAP-Funktion übernehmen und somit eine wichtige Rolle für die Dynamik oder Mobilität der Flagellen spielen. Das Hauptziel der im Falle des alpha19-Giardin durchgeführten Untersuchungen war die erste biochemische Charakterisierung des Proteins. Nach Bestätigung der Expression des alpha19-Giardins konnte mit Hilfe eines spezifischen Antikörpers das Protein ausschließlich in den ventralen Flagellen von G. lamblia nachgewiesen werden. Im Weiteren wurde für das alpha19 der typischen Annexin-Charakter sowie eine Phosphorylierung bestätigt. Das Vorkommen des alpha19-Giardins in der Membranfraktion des G. lamblia-Rohextraktes sowie die Solubilisierung des Proteins durch Zugabe eines Detergenz gaben erste Hinweise auf eine Fettsäuremodifikation des N-Terminus des Proteins, in dem ein Sequenzmotiv für eine Myristoylierung vorliegt. Folglich könnte das Protein an membrandynamischen Prozessen innerhalb der ventralen Flagellen beteiligt sein.

Der Intestinalparasit Entamoeba histolytica exprimiert während seines gesamten Lebenszyklus zwei Cysteinpeptidase-Inhibitoren der Chagasin-Familie (EhICP1 und EhICP2). Beide EhICPs inhibieren die peptidolytische Aktivität von E. histolytica-Zellextrakten und der humanen Peptidasen Cathepsin B und L unterschiedlich effektiv. Innerhalb der Trophozoiten von E. histolytica sind die EhICPs in verschiedenen Kompartimenten lokalisiert, EhICP1 im Cytosol und EhICP2 in Vesikeln, wo es mit verschiedenen lysosomalen Hydrolasen und mit phagocytierten Partikeln kolokalisiert. Im Vergleich zu den amöbialen Peptidasen liegen die EhICPs im molaren Unterschuss innerhalb der Zellen vor. Außerdem ist die Produktion und Lokalisationen der Peptidasen sowie der verschiedenen physiologische Prozesse, an denen die EhCPs beteiligt sind, unabhängig von der Expression der ehicp-Gene. Die erhaltenen Ergebnisse lassen darauf schließen, dass EhICP1 die Zelle vor versehentlich aus undicht gewordenen Lysosomen freigesetzten Peptidasen schützt, während EhICP2 an housekeeping-Prozessen, wie der Kontrolle der Prozessierung von Peptidasen, beteiligt sein könnte. Neben der Charakterisierung der Cysteinpeptidase-Inhibitoren aus E. histolytica bildeten Untersuchungen an einem Annexin-homologen Protein aus Giardia lamblia, einem anderen Intestinalparasiten, einen weiteren Schwerpunkt der hier vorgestellten Arbeit. Das Annexin-homologe alpha-19-Giardin nimmt unter allen Annexinen eine Sonderstellung dahingehend ein, als dass es als bisher einzig bekanntes Annexin ein N-terminales Signal für eine Doppelacylierung besitzt. Mit Hilfe von verschiedenen Expressionssystemen konnte hier experimentell belegt werden, dass alpha-19-Giardin tatsächlich als Substrat für eine Myristoyl- sowie für eine Palmitoyltransferase fungiert, und diese Modifikation die Membranassoziation des Proteins bewirkt.

No description available.

The development of a waterborne pathogen detection method that is rapid, multiplex, sensitive, and specific, would be of great assistance for water treatment facilities and would help protect water consumers from harmful pathogens. Here we have utilized surface enhanced Raman spectroscopy (SERS) in a sensitive multiplex pathogen detection method. Two strategies are proposed herein, one that utilizes SERS antibody labels and one that measures the intrinsic SERS signal of organisms. For the SERS label strategy, gold nanoparticles are conjugated with antibodies specific to Cryptosporidium parvum and Giardia lamblia and with organic dye molecules. The dye molecules, rhodamine B isothiocyanate (RBITC) and malachite green isothiocyanate (MGITC) were surface enhanced by the gold nanoparticles resulting in unique fingerprint SERS spectra. The SERS label method was successful in detecting G. lamblia and C. parvum simultaneously. The method was subsequently coupled with a filtration step to both concentrate and capture cysts on a flat surface for detection. Raman mapping across the filter membrane detected ~95% of the spiked cysts in the optimized system. In the second type of strategy, intrinsic virus SERS signals were detected with silver nanoparticles for enhancement. Principal component analysis performed on the spectra data set resulted in the successful differentiation of MS2 and PhiX174 species and also for the differentiation of viable virus samples and inactivated virus samples. / Ph. D.

Made available in DSpace on 2014-08-20T14:31:31Z (GMT). No. of bitstreams: 1 dissertacao_ana_berne.pdf: 2199093 bytes, checksum: 8e27f9fa1c22e061e919175c81aa8bb3 (MD5) Previous issue date: 2007-03-30 / The enteroparasitosis remains as an important public health problem in children in the Brazil, showing variable prevalence, according to the State and evaluated population. Studies with day-care center children are scarce, however, already knows that the exposure of the children in these places increased the susceptibility to parasitosis. Among the parasitosis the protozoary Giardia lamblia is responsible for severe diarrhea cases in children and the routine diagnosis methods presents many false negative results. The aim of this study was investigate the enteroparasitosis prevalence in children from a day-care public center of Rio Grande county, Rio Grande do Sul State and compare diagnosis techniques in samples of their fecal material to Giardia lamblia , the ELISA immunoassay and the centrifugal-sedimentation methods. 165 fecal samples where evaluated and processed by centrifugal-sedimentation and centrifugal-flotation methods, stained by trichromium and Kinyoun after the concentration by centrifugal-sedimentation. The general prevalence of enteroparasitosis was 64,2% (106/165). The most prevalent nematods species founded was Trichuris trichiura (24,2%) and Ascaris lumbricoides (22,4%) and the the most prevalent protozoary specie was Giardia lamblia (30,3%). The presence of opportunists coccids where also registered Cryptosporidium spp. (2,4%) and Isospora belli (0,6%). Among the positives 56,6% (60 samples) showed simple infection and 43,4% (46 samples) showed associated infection. The presence of non pathogenic protozoary like Entamoeba coli (15,2%), Endolimax nana (3,6%) and Enteromonas hominis (4,8%), indicated environmental fecal source contamination. The higher prevalence of nematods and protozoary in the studied population suggests the necessity of implementation of educational measures to prevent these enteroparasites. In the evaluation of comparative diagnosis of G. lamblia a higher positivity was verified in the ELISA technique 57% (90/158), followed by the centrifugal-sedimentation method 27,8% (44/158). The obtained results in this study suggests that is higher the prevalence of nematods and protozoary in the evaluated children and the ELISA technique to detect antigen in fecal sample showed higher efficiency to giardiasis diagnosis. / As enteroparasitoses ainda constituem um importante problema de saúde pública em crianças no Brasil, com prevalências bastante variáveis, conforme a região e população avaliada. Estudos com crianças que freqüentam creches são escassos, entretanto, sabe-se que nestes ambientes, as crianças estão mais expostas as parasitoses, dentre as quais o protozoário Giardia lamblia que é responsável por quadros graves de diarréia em crianças e os métodos de rotina utilizados no diagnóstico levam a muitos casos de falso-negativos. O objetivo deste trabalho foi investigar a prevalência de enteroparasitos em crianças de uma creche pública do Rio Grande, cidade portuária, localizada na região sul do estado do Rio Grande do Sul e comparar a técnica de ELISA (kit comercial Giardia II) com os métodos de centrífugo-flutuação e centrífugo- sedimentação para o diagnóstico de G. lamblia em fezes de crianças. Primeiramente foram avaliadas 165 amostras de fezes de processadas pelo método de centrífugo-sedimentação e centrífugo-flutuação e pelas colorações de tricrômio e de Kinyoun. A prevalência geral de enteroparasitos foi de 64,2% (106/165). Os nematódeos mais prevalentes foram Trichuris trichiura (24,2%) e Ascaris lumbricoides (22,4%) e o protozoário mais prevalente foi G. lamblia (30,3%). Também foram registradas as presenças dos coccídeos oportunistas Cryptosporidium (2,4%) e Isospora belli (0,6%). Dentre os positivos, 56,6% (60) apresentaram infecção simples e 43,4% (46) associadas. Constatou-se também a presença de protozoários não patogênicos, como Entamoeba coli (15,2%), Endolimax nana (3,6%) e Enteromonas hominis (4,8%), que indicou contaminação de origem fecal do ambiente. A alta prevalência de nematódeos e protozoários na população estudada sugere a necessidade de implementação de medidas educacionais, visando a prevenção destes enteroparasitos. Na avaliação do diagnóstico comparativo de G. lamblia foi verificado maior positividade para a técnica de ELISA, 57% (90/158), seguido do método de centrifugo-flutuação, 30,3% (48/158) e centrífugo-sedimentação, 27,8% (44/158). A partir dos resultados obtidos no presente estudo pode-se concluir que é alta a prevalência de nematódeos e protozoários nas crianças avaliadas e que a técnica de ELISA para detectar antígenos nas fezes é mais eficiente que os métodos de centrífugo-flutuação e centrifugo-sedimentação, podendo, portanto, ser utilizada, tanto no diagnóstico individual como em estudos epidemiológicos da giardíase.

Use of tubular anaerobic digesters to treat livestock waste in developing countries has energy, agricultural, health, social and environmental benefits. However, careful use of digester effluent as a soil amendment is required due to the potential presence of protozoan parasites Cryptosporidium parvum and Giardia lamblia. This research investigated the performance of four tubular digesters in the Monteverde region of Costa Rica. High (>75%) volatile solids and BOD5 removal efficiencies were observed, which was attributed to the formation of a biologically active floccular sludge layer. Computational fluid dynamics (CFD) and bioprocess models were developed to evaluate the transport and transformation mechanisms in the digesters. The CFD model estimated a mean liquid hydraulic residence time (HRT) of 23 days and the bioprocess model estimated an average mean cell residence time (MCRT) of 115 days. Cryptosporidium parvum and Giardia lamblia inactivation studies were performed in the laboratory under conditions similar to the environmental conditions observed in the field tubular digesters. The environmental conditions included: ambient temperatures (21-24°C), neutral pH and total ammonia nitrogen (TAN) concentrations below 250 mg NH4+-N/L. Inactivation rate constants for Cryptosporidium parvum and Giardia lamblia were 0.056 and 0.726 day-1, respectively. An (oo)cysts solid-liquid phase distribution study indicated that 70% of both (oo)cysts adhered to biosolids. A tubular digester model was used to estimate the concentration of viable (oo)cysts in the digester effluents. (Oo)cysts adhesion to solids, total solids concentration in the digester and HRT were the main factors contributing to the modeled effluent concentration of viable (oo)cysts. Since the model predicted presence of viable (oo)cysts in the tubular digester effluent, a quantitative microbial risk assessment (QMRA) model was developed to estimate the risk of infection from exposure to raw livestock waste and tubular digester effluents in two rural communities in Costa Rica. The risk of infection from Cryptosporidium parvum and Giardia lamblia was assessed for occupational and public exposure pathways; fomite and soil contamination and crop contamination from runoff. Results from the QMRA indicated that the concentration of (oo)cysts in the raw livestock waste, inactivation rates at the various exposure pathways and the treatment of livestock waste were the main contributing factors to the risk of infection. This research indicated that treatment of livestock waste in tubular digesters significantly decreased the risk of infection to below WHO’s acceptable individual annual risk of infection (10-4). This is the first study to combine mathematical modeling with field studies to determine the physical and biological processes in tubular digesters. This is also the first study to combine mathematical models with field and laboratory studies to determine the concentration of (oo)cysts in tubular digester effluents and to predict the risk of infection from Cryptosporidium parvum and Giardia lamblia if tubular digester effluent is used as a soil amendment.

Universiẗat, Diss.--Osnabrück, 1994.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-07-17T17:10:52Z No. of bitstreams: 1 Desenvolvimento da PCR em Tempo Real para.pdf: 2139383 bytes, checksum: 1075a6c994ede19cfbf82267ae9a76b9 (MD5) / Made available in DSpace on 2012-07-17T17:10:52Z (GMT). No. of bitstreams: 1 Desenvolvimento da PCR em Tempo Real para.pdf: 2139383 bytes, checksum: 1075a6c994ede19cfbf82267ae9a76b9 (MD5) Previous issue date: 2010 / CNPq e FAPESP / Fundação Oswaldo Cruz. Pavilhão Hélio Peggy e Pereira. Rio de Janeiro, RJ, Brasil / Giardia duodenalis (G. duodenalis) é um protozoário entérico patogênico distribuído mundialmente e apresenta amplo espectro de hospedeiros mamíferos, incluindo humanos. Isolados deste parasito possuem grande diversidade genética, apresentando sete genótipos (A-G) e diversos subtipos. No entanto, apenas os genótipos A e B infectam o homem e no subtipo A1 concentra-se o potencial zoonótico do parasito. Análise das sequências de amplicons de determinados marcadores moleculares demonstrou resultados discordantes na genotipagem de G. duodenalis evidenciando a necessidade de identificar novos marcadores. A proposta desse trabalho foi avaliar a aplicabilidade do locus da enzima málica (ME) comparando os resultados obtidos com o gene da β-giardina (βg) usando a PCR convencional seguida de sequenciamento para detecção e genotipagem de amostras clínicas. Foi desenvolvida uma PCR em Tempo Real - ME para detectar, quantificar e genotipar isolados de G. duodenalis diretamente de amostras de fezes. Foram usadas 100 amostras clínicas (83 humanas e 17 caninas) positivas segundo exame parasitológico e imunológico. A N - PCR convencional - βg amplificou 61% destas (52 amostras humanas e sete caninas) e todas foram caracterizadas como genótipo A, sendo 42 subtipo A1 (38 humanas e quatro caninas) e 19 subtipo A2 (16 humanos e três caninas). A N - PCR convencional - ME detectou apenas nove amostras (9%) positivas e todas pertencentes ao genótipo A, sendo seis humanas (quatro pertencentes ao subtipo A1, uma subtipo A2 e uma com subtipo indeterminado) e três caninas (todas A1). Ao correlacionar a genotipagem por ambos marcadores, observou-se concordância na identificação do genótipo. A comparação do limite de detecção da PCR convencional ME com a PCR em Tempo Real - ME demonstrou que esta última foi aproximadamente 10 4 vezes mais sensível. Análise estatística dos resultados obtidos com as réplicas da curva-padrão indicou reprodutibilidade do método e determinou que amostras negativas são aquelas com valores de Ct > 37. Desta forma, 71 amostras clínicas (71%) foram positivas na PCR em Tempo Real - ME com sonda para subtipo A1, destas 62 (87,3%) eram amostras humanas e nove (13,4%) caninas. A quantificação relativa de DNA de G. duodenalis nas amostras clínicas com a PCR em Tempo Real - ME, demonstrou que a concentração de cistos nas amostras analisadas variou de 4,21 ng a 204 fg (respectivamente, 22.000 e 1,0 cistos). A PCR em Tempo Real - ME apresentou maior sensibilidade em relação à N - PCR convencional - ME, indicando a necessidade de utilização de novos iniciadores, pois os utilizados encontram-se em regiões polimórficas, como demonstrado pela análise das sequências de ME. Apesar da necessidade de mais estudos, os resultados obtidos neste trabalho indicam que a PCR em Tempo Real - ME possui potencial aplicabilidade nos estudos de epidemiologia molecular da giardíase. / Giardia duodenalis (G. duodenalis) is an intestinal protozoan parasite found worldwide in various mammalian hosts, including humans. G. duodenalis isolates display high genetic diversity with seven genotypes (A-G) and subtypes. However, only A and B assemblages have been detected in humans. The subtype A1 parasite presents the strongest zoonotic potential. Discordant genotyping and detection results of G. duodenalis isolates have been previously reported using amplicon sequence analysis from certain molecular markers. Therefore, this leads to the search of novel ones. The purpose of this work was to compare conventional PCR, followed by sequencing, using malic enzyme (ME) and β-giardin gene (βg) as molecular markers for the detection and genotyping of the parasite found in faecal samples. Likewise, an approach involving Real Time PCR - ME for detecting, quantifying and genotyping G. duodenalis isolates was developed. We collected 100 positive faecal samples (83 humans and 17 canines) according to parasitological exam and immunoassays. N - PCR - βg detected 61 positive samples (61%) from which 52 were human and seven were canine. All those samples were characterized as genotype A. Subtype A1 and A2 were determined in 42 (38 humans/4 canines) and 19 (16 humans/3 canines) samples, respectively. However N - PCR - ME analyses revealed that only nine faecal samples (9%) were positive (6 humans/3 canines) for genotype A. Six human samples were subtyped as A1 (4), A2 (1) and one not-determined, and the three canine samples were subtype A2. However, when correlating the sample genotyping using both markers, we have observed an agreement in the identification of the genotypes. The comparison of the detection limit between the N - PCR - ME and the Real Time PCR - ME showed that the latter one was approximately 104-fold more sensitive. The statistical analysis of the data from the standard curve replicates indicated reproducibility of the method. Furthermore, Ct values > 37 were established as an indicative for negative samples. Therefore, Real Time PCR - ME analysis revealed that 71 samples (71%) were positive for subtype A1 from which 62 (87.3%) and nine (13.4%) samples were humans and canines, respectively. The relative quantification of G. duodenalis DNA in the clinical samples using Real Time PCR - ME varied from 4.21 ng to 204.0 fg corresponding to 22.000 and one cyst, respectively. Overall, Real Time PCR – ME analysis showed to be more sensitive than N - PCR - ME. It, thus, suggest the need to design different primers, because the ones used were within a polymorphic region of the ME sequence. Although more investigation is yet to be done, the results achieved in this work indicate that Real Time PCR - ME has a great potential in the applicability for molecular epidemiological studies of giardiasis.