O uso das águas costeiras para fins recreacionais está associado com benefícios à saúde e bem-estar, todavia eventuais impactos negativos podem diminuir estes benefícios. Esses usos variam de acordo com o tipo de atividade desenvolvida, sendo que a recreação de contato primário requer contato direto e prolongado com a água, durante a qual pode ocorrer ingestão acidental. A Resolução CONAMA nº 274/2000 dispõe sobre os critérios de balneabilidade e reza que as condições da qualidade das águas recreacionais devem ser avaliadas através de indicadores microbiológicos de contaminação fecal, e ainda recomenda que seja realizada pesquisa de organismos patogênicos em praias sistematicamente impróprias. Dada a escassez de dados da ocorrência de patógenos em águas costeiras, no período de 2010 a 2012, a CETESB realizou o Estudo de microrganismos patogênicos nas praias do Litoral Paulista pesquisando enterovírus, adenovírus, vírus da hepatite A, Cryptosporidium sp e Giardia sp, no intuito de preencher esta lacuna e gerar dados primários. Assim, o objetivo deste trabalho foi estimar a probabilidade de infecção por Cryptosporidium sp e Giardia sp após exposição a águas recreacionais costeiras usando como ferramenta a Avaliação Quantitativa de Risco Microbiológico (AQRM), como também o risco de doença. As concentrações de (oo)cistos nas águas das praias são oriundas dos relatórios de Qualidade das Praias Litorâneas no Estado de São Paulo da CETESB dos anos de 2011 e 2012. Nesse período foram analisadas 203 amostras coletadas de 12 praias na 1ª fase e de cinco praias na 2ª fase para a pesquisa de ocorrência de (oo)cistos. As amostras de água foram coletadas na isóbata de um metro, com frequência mensal. Giardia sp foi o microrganismo mais frequente, presente em 43 por cento das amostras e Cryptosporidium sp em 13 por cento . O cenário de exposição considerou tipos de atividade, tipos de usuários (crianças, adultos e esportistas), concentração de (oo)cistos, volume de ingestão, duração e frequência da exposição. A probabilidade de infecção foi maior em praias com mais amostras positivas para oocistos e cistos, no grupo dos esportistas e para Giardia sp. Em alguns casos os valores de risco de doença ultrapassaram o risco tolerável pela U.S. EPA (2012) de 3,6 por cento casos de gastroenterite, assim como ultrapassaram os resultados de incidência acumulada encontradas por LAMPARELLI et al. (2015). Os resultados apontaram a necessidade de melhoria nos sistemas de tratamento de efluentes no Litoral Paulista. A AQRM é uma ferramenta capaz de estimar a probabilidade de infecção no cenário das águas recreacionais e pode auxiliar no gerenciamento dos riscos. / The use of coastal water for recreational purposes has been associated with benefits to health and well-being; however some negative impacts can diminish such benefits. The usages can vary according to the type of activity but the primary contact demands physical contact resulting in a high probability in accidental ingestion of water. Brazilian legislation for coastal recreational waters CONAMA 274/2000 establishes criteria for fecal indicator bacteria and furthermore recommends investigation of pathogenic organisms for beaches which classification is systematically as improper. Given the scarcity of data referring to pathogenic presence in beaches´ waters, CETESB carried out a study, in 2010 and 2012, for quantifying enterovirus, adenovirus, hepatitis A virus, Cryptosporidium sp and Giardia sp in coastal waters of São Paulo state in order to obtain data about their occurrence of these pathogens in coastal waters. The objective of this study was to estimate the annual risk of infection and disease for Giardia sp and Cryptosporidium sp by ingestion of water during primary contact recreation using QMRA approach. Concentrations of both parasites were taken from the annual report entitled Quality of coastal beaches in São Paulo state by CETESB (2011 and 2012). In these years were analyzed 203 samples of water for quantifying (oo)cysts of Giardia and Cryptosporidium from 12 beaches in the first year and five beaches in the second year of research. The samples were collected at one meter isobaths, with monthly frequency. Giardia was the most frequent parasite present in 43 per cent of samples and Cryptosporidium sp in 13 per cent . Exposure scenario was built considering types of activity, beach goers (children, adults and athletes), concentration of parasites, ingestion rate, duration and frequency of exposure. The probability of annual infection was higher in beaches in which there were more positive results for parasites for athletes and for Giardia infection. The tolerable risk for gastroenteritis by USEPA, which is 3.6 per cent , was overpassed in some cases. Though the results found in this study overpassed the cumulative incidence reported by LAMPARELLI et al. (2015). The results indicate the need for improvements in wastewater treatment systems in the coastal area of São Paulo. As QMRA is a tool capable in estimating the probability of infection it can help to highlight crucial issues in risk management.

As doenças de veiculação hídrica, sobretudo aquelas causadas por protozoários intestinais, emergiram como um dos principais problemas de saúde pública. Diferentes aspectos são abordados sobre a biologia e a epidemiologia dos principais protozoários parasitas de transmissão hídrica. Cryptosporidium está descrito como um importante parasita associado a casos de surtos de veiculação hídrica e alimentos no mundo. A epidemiologia complexa desse protozóario e o fato de que a maioria das espécies e genótipos não pode ser diferenciada morfologicamente, aumentam o interesse por metodologias sensíveis e rápidas na detecção de espécies responsáveis pela infecção em humanos. Neste estudo foram avaliadas 50 amostras de água bruta superficial, coletadas no Rio São Lourenço da Serra (P1A) e Represa de Guarapiranga (P2A) e 50 de esgoto bruto coletadas em São Lourenço da Serra (P1E) e no poço vertical de Taboão da Serra (P2E) entre os meses de janeiro e dezembro de 2013. O isolamento dos oocistos na água foi realizado pelo Método 1623.1 e as amostras de esgoto bruto por centrifugação, separação imunomagnética (IMS). A caracterização genotípica ocorreu por meio da nested PCR, clonagem e sequenciamento com base no gene 18S rRNA comum a todas as espécies de Cryptosporidium. O ensaio de PCR em tempo real (qPCR) foi avaliado simultaneamente para detecção e quantificação de oocistos nas amostras. De acordo com os resultados obtidos pela nested PCR, Cryptosporidium foi detectado na água bruta superficial em 12 por cento (3/25) no manancial P1A e 16 por cento (4/25) no P2A. No esgoto bruto o parasito foi detectado em 20 por cento (5/25) das amostras no ponto P1E e 24 por cento (6/25) no poço vertical P2E. A qPCR detectou 52 por cento (0,79 a 1,85 oocistos/L) de amostras positivas no manancial P1A e o parasito foi detectado em 64 por cento (0,72 a 1,4 oocistos/L) no manancial P2A. No esgoto bruto 72 por cento das amostras foram positivas tanto no ponto P1E (7 a 655 oocistos/L) como no P2E (5 a 519 oocistos/L). A caracterização molecular permitiu a identificação de C. parvum e C. hominis na água bruta superficial, e C. hominis, C. parvum, e C. muris no esgoto bruto. As espécies do gênero Cryptosporidium identificadas neste estudo apresentam expressiva relevância para o desenvolvimento da doença humana. Neste sentido, as metodologias de concentração e caracterização empregadas nas análises demonstraram no geral, o potencial para aplicação em estudos de vigilância ambiental e foram úteis na diferenciação de espécies patogênicas. A presença de C. muris associada às espécies antroponóticas identificadas auxiliou na investigação de prováveis fontes de contaminação no ambiente confirmando a necessidade da expansão de medidas efetivas para proteção destes mananciais. / Waterborne diseases, especially those caused by intestinal protozoa, have emerged as a major public health problem. Different aspects are addressed on the biology and epidemiology of most waterborne protozoan parasites. Cryptosporidium is described as an important parasite associated with cases of waterborne and food outbreaks in the world. The complex epidemiology of this protozoan, as well as the fact that most species and genotypes cannot be differentiated morphologically, increase the interest for sensitive and rapid methods for the detection of species responsible for infection in humans. In this study, 50 samples of raw surface water were collected from São Lourenço River (P1A) and Guarapiranga Dam (P2A), and 50 samples of raw sewage were collected from São Lourenço da Serra (P1E) and from Taboão da Serras vertical well (P2E), between January and December of 2013. The isolation of oocysts in water was carried out by the USEPA Method 1623.1 and raw sewage samples were processed by centrifugation, immunomagnetic separation (IMS). Genotypic characterization occurred by nested PCR, cloning and sequencing based on 18S rRNA gene, which is common to all Cryptosporidium species. Real time PCR assays (qPCR) were carried out both for detection and quantification of oocysts simultaneously in the samples. According to the results obtained by nested PCR, Cryptosporidium was detected in raw surface water in 12 per cent (3/25) of samples at P1A and in 16 per cent (4/25) at P2A. In raw sewage the parasite was detected in 20 per cent (5/25) of samples at P1E and 24 per cent (6/25) in P2E vertical well. qPCR detected 52 per cent (0.79 to 1.85 oocysts/L) of positive samples at P1A, while the parasite was detected in 64 per cent (0.72 to 1.4 oocysts/L) of samples at P2A water supply. Regarding raw sewage, 72 per cent of samples were positive both at P1E (7 to 655 oocysts/L) and P2E (5 to 519 oocysts/L Molecular characterization allowed the identification of C. parvum and C. hominis in raw surface water, and C. hominis, C. parvum and C. muris in raw sewage. Cryptosporidium species identified herein belong to a group of organisms of significant relevance in waterborne diseases. Therefore, concentration and characterization methodologies applied in our analyses showed to be useful for environmental surveillance studies, as well as they were useful in the differentiation of human pathogens. The presence of C. muris associated to anthroponotic species helped in the investigation of likely contamination sources in the environment, confirming the need of expansion in effective measures to protect these water supplies.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-08-05T14:19:01Z No. of bitstreams: 1 2010 - Raquel Saucier Gomes.pdf: 7033294 bytes, checksum: d1538edd22d0ad5fcbf8c9c1e86f330e (MD5) / Made available in DSpace on 2016-08-05T14:19:01Z (GMT). No. of bitstreams: 1 2010 - Raquel Saucier Gomes.pdf: 7033294 bytes, checksum: d1538edd22d0ad5fcbf8c9c1e86f330e (MD5) Previous issue date: 2010-02-09 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico, CNPq. / This study aimed to diagnose and characterize genetically the species and genotypes of Cryptosporidium in stool samples from poultry and exotic birds marketed in Rio de Janeiro involving possible risk factors for infection. We analyzed 180 poultry sold in local markets and 103 exotic birds from breeding and pet shops. For analysis, the DNA extracted from fecal sample suspension was used to amplify the 18S rDNA sequences by nested-PCR technique. The amplicons generated were subjected to RFLP using the enzymes SspI and VspI, and sequencing and phylogenetic analysis, to confirm the species. Different species of Cryptosporidium were indentified in faecal samples of poultry and exotic birds. In the analysis of sites of enzymatic cutting products of nested-PCR gene 18SR DNA, the species C. bailey was the only one that displayed a characteristic cut-off, but other samples were. The following species were diagnosed: C. baileyi (GU082387) infecting ducks (Anas platyrhynchus domesticus), C. parvum in quails (Coturnix japonica) (GU082384 and GU082386), chicks (Gallus gallus domesticus) (GU082390 and GU082391), duck (GU082388) and Manon (Lonchura striata domestica) (GU074390). The Avian genotype III was identified in caulker (Lonchura padda oryzivora) (GUO74384) and cockatiel (Nymphicus hollandicus) (GU074385, GU074386 and GU074387). The sequences of canaries (Serinus canarius) received access numbers GU074388 and GU074389, but it was not possible to identify the species of Cryptosporidium, because of the large genetic distance between them and those already deposited in GenBank, suggesting a new genotype or a new specie. Although C. baileyi and Avian genotype III are common in birds, the diagnosis of C. parvum is a worrying finding, since this species are more associated with mammals. Birds can be considered as reservoirs and disseminators of environmental infective form of the parasite, allowing the infection to a large number of species of hosts, including in the man in the epidemiological chain. / O presente trabalho teve por objetivo diagnosticar e caracterizar geneticamente esp?cies e/ou gen?tipos de Cryptosporidium em amostras fecais de aves dom?sticas e ex?ticas comercializadas no Estado do Rio de Janeiro, associando poss?veis fatores de risco da infec??o. Foram analisadas 180 aves dom?sticas comercializadas em mercados locais e 103 aves ex?ticas de criadouros e petshops. Para as an?lises, o DNA extra?do de suspens?o de amostra fecal foi utilizado na amplifica??o das seq??ncias do 18S rDNA atrav?s da t?cnica Nested-PCR. Os amplicons gerados foram submetidos ? RFLP, utilizando as enzimas SspI e VspI, e ao seq??nciamento, para a confirma??o das esp?cies. Foram identificadas esp?cies de Cryptosporidium em amostras fecais de aves dom?sticas e ex?ticas. Durante as an?lises dos s?tios de corte enzim?tico dos produtos da Nested-PCR do gene 18Sr DNA, a esp?cie C. baileyi foi a ?nica que apresentou padr?o de corte caracter?stico, por?m nas demais amostras foi necess?ria a confirma??o atrav?s do sequenciamento e estudo filogen?tico. Foi diagnosticado C. baileyi (GU082387) infectando patos (Anas platyrhynchus domesticus); C. parvum em codornas (Coturnix coturnix japonica) (GU082384 e GU082386), pintos (Gallus gallus domesticus) (GU082390 e GU082391), pato (GU082388) e manon (Lonchura striata domestica) (GU074390). O gen?tipo avi?rio III foi identificado pela primeira vez em calafate (Lonchura padda oryzivora) (GUO74384) e em calopsita (Nymphicus hollandicus) (GU074385, GU074386 e GU074387). As seq??ncias de can?rios (Serinus canarius) receberam os n?meros de acesso GU074388 e GU074389, por?m n?o foi poss?vel a identifica??o da esp?cie de Cryptosporidium, devido ? grande dist?ncia gen?tica entre elas e aquelas j? depositadas no GenBank, sugerindo novo gen?tipo ou uma nova esp?cie . Embora C. baileyi e o gen?tipo Avi?rio III sejam comuns em aves, o diagn?stico de C. parvum ? um achado preocupante, j? que esta esp?cie est? mais associada com mam?feros. Aves podem ser consideradas como reservat?rios e disseminadoras ambientais da forma infectante do protozo?rio, possibilitando a infec??o para um amplo n?mero de esp?cie de hospedeiros incluindo, nesta cadeia epidemiol?gica, o ser humano.

Este trabalho teve por objetivo estudar a desinfecção seqüencial cloro-radiação ultravioleta e ozônio-radiação ultravioleta a fim de avaliar a resistência de microrganismos indicadores de poluição fecal - E. coli, coliformes totais e Clostridium perfringens - e compará-la à dos protozoários patogênicos: Giardia spp. e Cryptosporidium spp.. Houve, primeiramente, monitoramento do reator anaeróbio UASB cujo efluente foi utilizado neste estudo. Foi, então, verificado o comportamento das espécies residuais de cloro no esgoto e conduzido um estudo de parâmetros indicadores (pH, potencial de oxi-redução e condutividade) para o breakpoint. Na desinfecção, foram empregados o cloro, ozônio e radiação ultravioleta; separados, e seqüencialmente. Os ensaios realizados com ozônio promoveram remoção de DQO, sólidos e dos valores de absorbância em 254 nm, diferentemente do cloro. A ordem crescente de resistência à desinfecção foi: E. coli < coliformes totais < C. perfringens < protozoários. Houve correlação em alguns ensaios entre a bactéria esporulada C. perfringens e Giardia spp.. O efeito sinérgico, promovido pela desinfecção seqüencial, foi evidenciado em alguns experimentos para C. perfringens e Giardia spp. / The present dissertation reports on the study of the sequential disinfection with chlorine-ultraviolet radiation and ozone-ultraviolet radiation to evaluate the resistance of microorganisms that indicate fecal pollution - E. coli, total coliforms and Clostridium perfringens - and compare it to the resistance of pathogenic protozoa: Giardia spp. and Cryptosporidium spp.. Firstly, the UASB reactor, whose effluent was utilized in this study, was monitored. Then the behavior of the residual species of chlorine in the sanitary sewage was verified and a study of the indicating parameters (pH, oxi-reduction potential and conductivity) for the breakpoint was conducted. Chlorine, ozone and ultraviolet radiation were used separately and sequentially in the disinfection. Tests performed with ozone promoted removal of COD, solids and absorbance values of 254 nm, differently from chlorine. The order of resistance to disinfection was: E. coli < total coliforms < Clostridium perfringens < protozoa. There was a correlation in some tests between spore-forming bacterium Clostridium perfringens and Giardia spp.. The synergic effect caused by the sequential disinfection was observed in some experiments for C. perfringens and Giardia spp.

Le genre Cryptosporidium (Apicomplexa : Alveolata) comprend des espèces qui infectent l'intestin d'un grand nombre de vertébrés (l'homme compris). Elles sont la cause de la cryptosporidiose, maladie opportuniste émergente avec un impact considérable chez le patient immunodéficient, notamment sidéen. Ces protistes infectent aussi des sujets immunocompétents dans toutes les latitudes, en déterminant des diarrhées en général autorésolutives. Les oocystes hébergeant les sporozoïtes infectants sont éliminés avec les selles des hôtes infectés, contaminent l'environnement, sont fréquemment véhiculés par les eaux où ils gardent leur pouvoir infectieux pendant longtemps, résistant aux désinfectants usuels. Par ailleurs, étant immédiatement infectieux après leur excrétion, ils peuvent être transmis directement par contact inter-humain.<br />Sans prescription spécifique, rare dans les faits, la détection d'oocystes de Cryptosporidium n'est pas pratiquée lors de l'examen coproparasitaire conventionnel. De plus, la morphologie étant insuffisante à la distinction des espèces dans le genre, leur identification, qui fait appel à des méthodes moléculaires, est rarement pratiquée, notamment dans les pays en développement. Cependant, elle constitue le seul moyen de déterminer les sources, les voies et les mécanismes de l'infection, informations essentielles au développement de stratégies rationnelles de prévention.<br />Pour toutes ces raisons, nous avons dans un premier temps cherché à caractériser la variabilité génétique des espèces et de sub-espèces de Cryptosporidium dans différentes régions : Venezuela, Francia, Haïti e Iran. Au Venezuela, chez les 397 patients avec un statut VIH/SIDA confirmé, notre étude a révélé que l'infection par Cryptosporidium est fréquente parmi les patients infectés par le VIH vivant à Caracas, que l'infection, dont la prévalence augmente avec l'âge, s'associe fréquemment à une diarrhée (plus de 5 selles par jour) et à une perte de poids, et qu'un taux de CD4+ <100 cell/mm3 peut être considéré comme un facteur de risque prédictif de cryptosporidiose. L'étude génotypique (PCR-RFLP ciblant l'ADNr 18S de Cryptosporidium sp) a permis d'identifier les espèces isolées ; le génotypage multiloci, qui ciblait des mini- et microsatellites a autorisé une résolution à niveau infra-spécifique. Trois espèces de Cryptosporidium ont été identifiées parmi des nouveaux échantillons fécaux de patients vénézuéliens VIH+: C. hominis, C. parvum et C. canis. L'analyse avec les marqueurs mini- et microsatellites a révélé que les échantillons de C. hominis présentaient une même combinaison d'allèles. Nous avons effectué des études semblables en France, en Haiti et en Iran. Globalement, la structure génétique des populations de ces parasites montre une prédominance clonale, malgré l'existence d'un stade sexuel obligatoire dans le cycle biologique des Cryptosporidium spp. L'origine géographique et l'espèce hôte ont un rôle structurant. En Iran, trois espèces de Cryptosporidium ont été identifiées parmi les échantillons fécaux de 15 personnes et 9 animaux: C. parvum, C. hominis et C. meleagridis.<br />La seconde partie de ce travail décrit le développement d'un modèle murin immunodéprimé, représenté par des souris SCID (Severe Combined Immunodeficiency), traitées par la dexaméthasone. Ce modèle, destiné à caractériser les isolats de Cryptosporidium spp sur le plan phénotypique, a révélé des divergences marquées entre C. parvum et C. muris. Les espèces C. hominis, spécifique de l'homme, et C. molnari, de poisson, ne se sont pas développées chez la souris SCID traitée ou pas par la déxaméthasone. L'étude histopathologique a confirmé la localisation gastrique (préférentiellement fundique) de C. muris. Les souris SCID sous dexaméthasone inoculées avec C. parvum ont développé des dysplasies iléo-caecales à partir du 35ème jour post-inoculation. Chez les souris non traitées par la déxamethasone euthanasiées 57 jours après l'infection la parasitose était associée à un néoplasie intestinal de bas grade. Des néoplasies de haut grade ont été observés au niveau de l'estomac (souris sous déxaméthasone), principalement dans la zone antrale. Globalement, nos expériences ont montré que C. parvum induit des néoplasies intra-épithéliales de bas et de haut grade dans l'estomac, le duodénum, le caecum et autres régions du côlon. Un tiers de ces souris présentaient des lésions néoplasiques dans plus d'un type d'organe. Des approches immunohistochimiques et biochimiques complètent la caractérisation de ces lésions.<br />Globalement, ce travail, qui rapporte un modèle expérimental hautement reproductible de cryptosporidiose, fournit des nouvelles informations sur la diversité génétique de Cryptosporidium dans plusieurs régions du monde et sur les différences biologiques entre espèces. En particulier, ce travail démontre pour la première fois la capacité de C. parvum à induire des processus néoplasique chez l'hôte. Cette découverte majeure accroît significativement l'intérêt scientifique des Cryptosporidium spp et suggère que l'impact de ces parasites en santé humaine et animale pourrait être beaucoup plus grand que ce qu'on croî

In den vorliegenden Studien wurden verschiedene diagnostische Verfahren zum Nachweis von Protozoen und Nematoden im Hinblick auf Sensitivität, Arbeitsaufwand und Kosten miteinander verglichen. Zudem wurde die Eignung der PCR zur molekularen Charakterisierung der Cryptosporidium spp. exemplarisch an Igelkotproben getestet. Bei der Untersuchung von 90 Ferkelkotproben auf I. suis war die Sensitivität eines Kotausstriches mit nachfolgender Autofluoreszenzmikroskopie (AM) signifikant höher als bei einem Flotationsverfahren (FV) mit NaCl-Zucker-Lösung und bei dem kombinierten Sedimentations-Flotations-Verfahren (KSFV) mit verschiedenen Flotationslösungen (NaCl, ZnSO4, NaCl-Zucker-Lösung) mit nachfolgender Lichtmikroskopie. Zudem ist der Arbeitsaufwand für die AM deutlich geringer als bei dem FV und KSFV. Die höheren apparativen Kosten für die AM sind bei hohem Probendurchsatz durch den geringeren Zeitaufwand und der höheren Sensitivität gerechtfertigt. Die Anzahl Kryptosporidien-positiver Proben war bei der Untersuchung von 103 Kälberkotproben auf Cryptosporidium sp. mittels Enzymimmunoassays (EIA; ProSpecT® Cryptosporidium Microplate Assay) im Vergleich zur Karbolfuchsin-Färbung (CF) nach HEINE (1981) und der modifizierten-Ziehl-Neelsen-Färbung (MZN) nach HENRIKSEN u. POHLENZ (1982) am höchsten und signifikant höher als bei der Anwendung der MZN, wenn 10 Blickfelder durchmustert wurden. Bei der Untersuchung von 74 Igelkotproben auf Cryptosporidium sp. mittels EIA (ProSpecT®), einem immunochromatographischen Verfahren (FASTest® CRYPTO Strip), der MZN nach HENRIKSEN u. POHLENZ (1981) und einem direkten Immunfluoreszenz-Test (IFA; MERIFLUOR Cryptosporidium/Giardia) wurden in 9 (EIA), 10 (FASTest®), 11 (MZN) und 12 (IFA) Proben Cryptosporidium sp. nachgewiesen. Der Arbeitsaufwand des FASTest® und der CF ist mit dem EIA vergleichbar, während der IFA und die MZN mehr Zeit benötigen. Die Anwendung des FASTest®, des IFA und des EIA ist mit höheren Kosten verbunden als bei den Färbemethoden, können aber gut in den Arbeitsablauf eines diagnostischen Labors eingefügt werden und sind einfach auszuwerten. Darüber hinaus wurden 45 Kotproben, welche bis zu 27 Tage bei verschiedenen Temperaturen (+6 °C, +16 °C, +30 °C, +40 °C) gelagert wurden, untersucht, um einen Einfluss der Temperatur auf das Untersuchungsergebnis von EIA, CF und MZN zu ermitteln. Während sich die Anzahl positiver Proben bei der Untersuchung mit den Färbemethoden temperatur- und zeitabhängig reduzierte, wurde das Untersuchungsergebnis mittels EIA von der Lagerungstemperatur nicht beeinflusst, so dass ungekühlt transportierte Proben vorzugsweise mit dem EIA untersucht werden sollten. Dagegen ist die CF aufgrund ihrer einfachen und preiswerten Durchführung zur Untersuchung einer hohen Anzahl an Proben geeignet, sofern eine ununterbrochene Kühlung der Proben gewährleistet ist und diese innerhalb von drei Tagen untersucht werden. Der FASTest® ist zur Anwendung in Tierarztpraxen und Ställen geeignet, da zur Untersuchung kein Mikroskop benötigt wird und die Resultate schnell vorliegen. Die Verwendung des IFA, der Kryptosporidien-Oozysten und Giardien-Zysten nachweist, bietet sich vor allem bei Proben an, die auf beide Protozoen untersucht werden sollen. Das Vorkommen der Kryptosporidiose bei unterentwickelten und geschwächten Igeln, welche zum Überwintern in Igelstationen aufgenommen werden, ist hoch. Von 188 untersuchten Igelkotproben konnten in 29,8 % der Proben Cryptosporidium spp. nachgewiesen werden. Durch die Genotypisierung der Kryptosporidien aus 15 positiven Igelkotproben mittels RFLP-PCR basierend auf dem 18S rRNA-Gen konnte in allen untersuchten Proben die Präsenz von C. parvum gezeigt werden. Mit Hilfe der Multilocus-Sequenz-Typisierung der Fragmente des 60kDa Glycoprotein-Gens, des 18S rRNA-Gens, des Actin-Gens und des 70 kDa Hitzeschockprotein-Gens konnten drei verschiedene Subtypen-Familien (IIa, IIc und eine neue als VIIa vorgeschlagene Subtypen-Familie) erkannt werden. Die von den Igeln ausgeschiedenen Kryptosporidien-Oozysten mit zum Teil nachgewiesenem zoonotischen Potential (IIa Subtypen-Familie) könnten eine Infektionsquelle für den Menschen sein, aber auch ein antropozoonotisches Potential (IIc Subtypen-Familie) sollte in Betracht gezogen werden, so dass die Hygiene in den Igelstationen einen hohen Stellenwert einnehmen sollte. Die Untersuchungsergebnisse zum Nachweis von Eimeria-Arten beim Kalb von 70 Sammelkotproben, hergestellt aus 10 Einzelkotproben (SKP10), bzw. von 30 Sammelkotproben, zusammengesetzt aus 5 Einzelkotproben (SKP5), wurden mit denen der zugehörigen Einzelkotproben (EKP) verglichen. Die Resultate der EKP (arithmetischer Mittelwert) und der zugehörigen SKP weisen mit den signifikant häufigeren Abweichungen im Bereich von bis zu 100 Oozysten pro Gramm Kot (OpG) eine geringe Differenz zwischen den beiden Verfahren auf. Durch den sicheren Nachweis von Eimeria-Oozysten bei einem erwarteten Oozystengehalt von nur 202 OpG (SKP10) und 122 OpG (SKP5) ist die Untersuchung von Kälbersammelkotproben, eine Methode mit geringem Arbeitsaufwand und geringen Untersuchungskosten, zum Nachweis einer klinischen oder subklinischen Kokzidiose geeignet. Bei 51 Pferdekotproben wurde jeweils dreimal das kombinierte Sedimentations-Flotations-Verfahren (KSFV), wobei die Entnahme von verschiedenen Lokalisationen der Kotprobe (aus der Randregion, dem Inneren oder aus beiden Lokalisationen) erfolgte, und jeweils dreimal das KSFV mit vorheriger Homogenisierung einer größeren Kotmenge zum Nachweis von Nematodeneier durchgeführt. Eine Anhäufung der Strongyliden- und Ascarideneier in einem bestimmten Bereich der Proben konnte durch die Untersuchungen der verschiedenen Lokalisationen (á 10 g Kot) nicht nachgewiesen werden, so dass eine weitgehend homogene Verteilung dieser Nematodeneier in einer Pferdekotprobe wahrscheinlich ist. Zudem konnten die Untersuchungsergebnisse des KSFV, bei welchem 10 g Kot untersucht werden, durch die vorherige Homogenisierung einer größeren Probenmenge nicht verbessert werden. Zum Nachweis von Nematoden beim Pferd sollte dem Labor eine ausreichende Probenmenge (ca. 50 g) zugesandt werden. Die Homogenisierung einer größeren Probenmenge vor der Durchführung einer diagnostischen Methode, bei der Aliquote von mindestens 10 g Kot Verwendung finden, ist unnötig. / The studies presented were carried out to compare different diagnostic methods for detection of protozoa and nematodes regarding sensitivity, expenditure of human labour and costs. Besides, the ability of the PCR for the molecular characterization of the Cryptosporidium spp. was tested exemplarily in faecal samples of hegdehogs. The examination of ninety faecal samples of suckling piglets showed a significantly higher sensitivity of faecal smears examined by autofluorescence microscopy (AM) compared to the flotation method (FV) using NaCl-sucrose solution and the combined sedimentation-flotation method (KSFV) using different flotation solutions (NaCl, ZnSO4, NaCl-sucrose) scanned by bright field microscopy. Moreover the expenditure of human labour by AM is considerably lower than FV and KSFV. The costs related to equipment for AM is justified in case of high sample throughput and by superior sensitivity. The enzyme immunoassay (EIA; ProSpecT® Cryptosporidium Microplate Assay) was the most sensitive method for diagnosis of cryptosporidiosis in calves (n = 103) compared to the carbol fuchsin (CF; HEINE 1981) and modified Ziehl-Neelsen (MZN; HENRIKSEN a. POHLENZ 1982) staining techniques. The sensitivity of the EIA was significantly higher than the MZN, if ten fields of view were scanned. 74 faecal samples of hedgehogs were examined with the EIA (ProSpecT®), an immunochromatographic method (FASTest® CRYPTO Strip), the MZN (HENRIKSEN u. POHLENZ (1981)) and a direct immunofluorescent assay (IFA; MERIFLUOR Cryptosporidium/Giardia). Cryptosporidium sp. were detected in 9 (EIA), 10 (FASTest®), 11 (MZN) und 12 (IFA) faecal samples. The hands on time of the FASTest® and CF is comparable to EIA while the IFA and MZN are more time-consuming. The examination of the FASTest®, IFA and EIA is combined with higher costs than the staining techniques, but they can be integrated in the work flow of a routine diagnostic laboratory easily and evaluation is simple. Moreover 45 faecal samples stored up to 27 days at different temperature (+6 °C, +16 °C, +30 °C, +40 °C) were examined to evaluate the influence of temperature on the results of EIA, CF and MZN. While the number of the positive samples of stained smears decreased in a temperature and time-dependent manner, the results of the EIA were not influenced by sample storage at any temperature, so that samples transported without cooling should be examined preferably by EIA. Nevertheless the CF due to its simplicity and low costs is suited for scanning of a high number of samples, if they were cooled continuously and examined within three days. The FASTest® is qualified for use in veterinary practice and stables, because the examination requires no microscope and the results are obtained immediately. The IFA, which can detect Crypotsporidium oocysts as well as Giardia cysts, is suited especially for faecal samples suspected to contain both protozoa. Cryptosporidial infections are very frequent in hedgehogs which are admitted for hibernation to hedgehog rehabilitation centres because of their insufficient body weight and weakness. Cryptosporidium spp. were detected in 29.8 % of 188 faecal samples of hedgehogs. The genotyping of Cryptosporidium spp. by PCR and RFLP-PCR based on the 18S ribosomal RNA gene were performed on 15 faecal samples of hedgehogs positive for Cryptosporidium spp. and suggested the presence of C. parvum in all samples. Multilocus sequence typing on partial 60 kDa glycoprotein gene, 18S rRNA gene, actine gene, 70 kDa heat shock protein gene sequences revealed 3 different subtype families: IIa, IIc and a new proposed as VIIa subtype family. Some of the Cryptosporidium oocysts excreted from hedgehogs are zoonotical (IIa subtype family) or anthropozoonotic(IIc subtype family). Thus hygienic measurements to avoid transmission are essential in hedgehog rehabilitation centres. The results of examination of 70 pooled faecal samples originating from 10 calves (SKP10) and 30 pooled faecal samples originating from 5 calves (SKP5) for detection of Eimeria spp. were compared with the arithmetic means of opg (oocysts per gram of faeces) counts of the respective single 10 or 5 samples. A low difference between both methods of less than 100 opg was significantly more frequently observed than higher differences. Low values of 202 opg and 122 opg were reliably detected in SKP10 und SKP5, respectively, and thus examination of pooled faecal samples appears to be suitably sensitive and cost effective to detect clinical and subclinical coccidiosis in calves. 51 faecal samples of horses were examined three times by KSFV for nematode eggs by taking aliquots from different locations of the same faecal samples (from the margin, from inside and from both locations). Thereafter the KSFV with the homogenisation of a larger amount of faeces was also carried out three times. The examination of samples from the different locations (each 10 g of faeces) delivered no evidence for accumulation of nematode eggs (strongyles and Parascaris equorum) in the faeces and thus the distribution of the nematode eggs appears sufficiently homogeneous in faecal samples of horses. Homogenisation of a larger amount of faeces did not improve the results of coproscopy. For diagnostic purposes 50 g faeces per sample should be shipped to the laboratory. The homogenisation of a larger amount of faeces before using a diagnostic method is dispensable, if aliquots of 10 g faeces are examined.

Title from first page of PDF document. Includes bibliographical references.

Die Kryptosporidiose stellt aufgrund ihres zoonotischen Charakters und der Entwicklung chronischer Durchfälle bei Immunsupprimierten ein hohes Gesundheitsrisiko für den Menschen, aber ebenso für Tiere dar. Derzeit verfügbare Therapeutika ermöglichen keine zuverlässige Bekämpfung klinischer Symptome oder eine Erregerelimination, daher ist die Erforschung neuer Therapieansätze dringend notwendig. CDPK stellen in diesem Zusammenhang interessante Zielmoleküle dar, da sie zwar in Pflanzen und Protisten einschließlich Apikomplexa, jedoch nicht in Pilzen und Säugetieren vorkommen. Trotz der Entdeckung vielversprechender neuer Wirkstoffe gegen CpCDPK1 in den letzten Jahren ist zur Lokalisation und Funktion von CDPK in C. parvum wenig bekannt.Diese Arbeit belegt die Transkription von sechs CpCDPK in vitro und beschreibt erstmals die Länge der 3’UTR von CpCDPK. Die Translation wurde durch den Nachweis spezifischen Proteins in Sporozoiten im Immunoblot sowie die Lokalisation von CpCDPK1 mit Hilfe der Immunfluoreszenz belegt. Möglicherweise wird die CpCDPK1 durch N-Myristoylierung an Membranen gebunden, an die Oberfläche von Zoiten gebracht und sezerniert. Eine Rolle des Enzyms im Invasions- und Egressmechanismus des Parasiten wird diskutiert.

The development of immune response accountable for the ability to control Cryptosporidium muris TS03 infection was studied using immunocompetent and various types of immunodeficient mouse models. Subsequently the immune response was characterized by analysis of leukocyte infiltration and cytokine production in gastric epithelium. Moreover, the potentiality of immunocompetent mice to develop effective immune response to C. andersoni LI03 infection with consequent protection to consequent infection of the same mice with C. muris TS03 was also studied by monitoring oocysts shedding, leukocyte infiltration of the gastric mucosa and cytokine production in ex vivo cultures of splenocytes.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O Cryptosporidium é um parasito coccídeo reconhecido por causar diarréia em humanos e animais em todo o mundo. O gênero compreende pelo menos 20 espécies confirmadas, sendo o C. hominis e C. parvum as principais espécies causadoras de criptosporidiose em humanos. Ferramentas moleculares têm sido desenvolvidas para detectar e diferenciar espécies/genótipos e subgenótipos de Cryptosporidium. O objetivo do trabalho foi avaliar a heterogeidade molecular de Cryptosporidium sp. obtidos de amostras clínicas provenientes dos municípios do Rio de Janeiro e de Salvador, através da PCR em tempo real e seqüenciamento automático. Foram analisadas 85 amostras, distribuídas em 3 grupos distintos, sendo 45 delas do município do Rio de Janeiro e 40 amostras provenientes de Salvador, Bahia. Todas as amostras foram positivas para Cryptosporidium sp. pelo método de coloração de Kinyoun a frio. O ensaio da PCR em tempo real combinou uma reação multiplex para a detecção do gênero Cryptosporidium e da espécie C. parvum e uma reação simples para a detecção de C. hominis. Na detecção do gênero Cryptosporidium foram utilizados par de primers e uma sonda TaqMan desenhados a partir do alinhamento de seqüências conservadas do gene 18S rRNA de várias espécies de Cryptosporidium disponíveis no GenBank. Para a detecção das espécies C. parvum e C. hominis foram utilizados primers e sondas específicos obtidos a partir de seqüências de cada espécie disponíveis no GenBank. A detecção do gênero Cryptosporidium através da sonda 18S rRNA, na reação duplex, foi visualizada em 63 de 85 amostras totais. Destas, a sonda TaqMan específica para C. parvum detectou 6 amostras e a sonda TaqMan específica para C. hominis detectou 42 amostras. Quinze amostras não puderam ser detectadas pelas sondas C. hominis ou C. parvum. Nos ensaios da PCR para o gene 18S, 31 amostras foram positivas e 27 delas sequenciadas. As análises filogenéticas confirmaram a presença de C. hominis, C. parvum e C. felis nas amostras estudadas. Na análise da topologia da árvore filogenética obtida por Neighbor Joining, observou-se-se que as sequências obtidas neste estudo se agruparam com espécies do gênero Cryptosporidium já descritas, com 99% ou 100% de similaridade. Conclui-se que os dois métodos utilizados são importantes ferramentas para o diagnóstico da criptosporidiose e diferenciação de C. hominis e C. parvum em investigações epidemiológicas, assim como avaliação da heterogeneidade molecular de Cryptosporidium sp / Cryptosporidium is a coccidia parasite known to cause diarrhea in humans and animals worldwide. The genus comprises at least 20 confirmed species, with C. hominis and C.parvum major species that cause cryptosporidiosis in humans. Molecular tools have been developed to detect and differentiate Cryptosporidium at the species/genotypes and subtype levels. The objective of this study was to evaluate the molecular heterogeneity of Cryptosporidium sp. clinical samples obtained from Rio de Janeiro and Salvador (Bahia), through real-time PCR and automatic sequencing. We analyzed 85 samples, distributed in three distinct groups, 45 of them in the city of Rio de Janeiro and 40 samples from Salvador. All samples were positive for Cryptosporidium sp. by modified Kinyoun acid-fast staining technique. The real-time PCR procedure combined a duplex reaction for the detection of Cryptosporidium species and C. parvum and a simple reaction for the detection of C. hominis. The detection of the genus Cryptosporidium have been used a pair of primers and TaqMan probe designed from the alignment of conserved sequences of the 18S rRNA gene of several species of Cryptosporidium available in GenBank. For the detection of species C. parvum and C. hominis were used specific primers and probes derived from sequences of each species available in the GenBank. Detection of Cryptosporidium sp. by 18S rRNA probe, in the duplex reaction, was visualized in 63 of 85 total samples. Of these, the C. parvum TaqMan probe detected 6 samples and the C. hominis TaqMan probe detected 42 samples. Fifteen samples could not be detected by C. hominis or C. parvum probes. In the 18S PCR assays, 31 samples were positive and 27 of them sequenced. Phylogenetic analysis confirmed the presence of C. hominis, C. parvum and C. felis. The analysis of the phylogenetic tree obtained by Neighbor Joining showed that the sequences obtained in this study were grouped with Cryptosporidium species described in the GenBank, with 99% or 100% similarity. Both methods are important tools for the diagnosis of cryptosporidiosis and differentiation of C. hominis and C. parvum in epidemiological investigations, as well as evaluation of Cryptosporidium sp. molecular heterogeneity