Esgotos urbanos constituem compartimentos ambientais que favorecem a emergência e re-emergência enfermidades de veiculação hídrica. Este estudo objetivou analisar cistos de Giardia spp., oocistos de Cryptosporidium spp., assim como cistos, ovos e larvas de outros protozoários e helmintos patogênicos; Coliformes Totais e Termotolerantes, Rotavírus e Adenovírus em amostras de esgoto bruto e tratado da Estação de Tratamento de Esgoto de Ribeirão Preto - SP. As coletas de esgoto foram realizadas no ponto de entrada e no ponto de saída da ETE-Ribeirão Preto/SP. As análises de cistos de Giardia spp. e oocistos de Cryptosporidium spp. foram realizados pelo Método 1623 da EPA. Ovos, cistos e larvas de outros protozoários e helmintos foram analisados pelo Método de Sedimentação proposto pela CETESB (1989). A análise de coliformes totais e termotolerantes foi realizada pela Técnica de Tubos Múltiplos (Colilert®). A análise de vírus foi realizada através do Teste de Elisa com o KIT da RIDASCREEN® Enzimaimunoensaio. Os resultados obtidos revelaram que a concentração de Giardia spp. no esgoto bruto variou de 120 a 2200 cistos/L, já no esgoto tratado essa concentração variou de 0,45 a 3,5 cistos/L. Com relação aos oocistos de Cryptosporidium spp. a concentração no esgoto bruto variou de não detectável a 28,9 oocistos/L e no esgoto tratado as concentrações variaram de não detectável a 1,05 oocistos/L. O processo de lodos ativados na ETE-RP promoveu uma remoção parcial de parasitas, tais como: Endolimax nana, Entamoeba coli, Entamoeba hystolitica, Ancylostoma sp., Ascaris sp. Fasciola hepatica e Strongyloides stercoralis, cujo fator de redução variou entre 18,2 e 100%. Pode-se observar que houve diferença estatisticamente significante entre o número mais provável de coliformes totais e termotolerantes no esgoto bruto e esgoto tratado, mostrando uma redução na concentração desses organismos indicadores após o tratamento do esgoto na ETE-Ribeirão Preto/SP. Não houve correlação significativa entre as concentrações de coliformes totais e termotolerantes em relação à concentração de cistos de Giardia spp. e oocistos de Cryptosporidium spp.. A análise de vírus mostrou resposta positiva para Rotavirus e Adenovirus em todas as amostras analisadas (esgoto bruto e tratado), mostrando menor absorvância no esgoto tratado. Os resultados obtidos contribuem para a identificação e caracterização de microorganismos de veiculação hídrica no esgoto tratado na ETE-RP, fornecendo dados úteis para a definição de políticas públicas de saneamento, referentes ao controle da qualidade microbiológica do esgoto no país. / Sewage treatment is still precarious in many Brazilian cities, which has contributed to the emergence and re-emergence of waterborne diseases. This study analyzed Giardia spp. cysts and Cryptosporidium spp. oocysts, as well as cysts, eggs and larvae of other protozoa and helminthes. Also part of the study were total and thermotolerant coliforms, Rotavirus and Adenovirus in samples of raw and treated sewage of the Sewage Treatment Station (ETE) of Ribeirão Preto, SP, Brazil. Sewage samples were taken at the points of entry and exit of the ETE, Ribeirão Preto. Analyses of Giardia spp. cysts and Cryptosporidium spp. oocysts were performed through the EPA\'s method 1623. Eggs, cysts and larvae of other protozoa and helminthes were analyzed utilizing the method of sedimentation as proposed by CETESB (1989). Analyses of total and thermotolerant coliforms were performed using the Multiple Tube method (Colilert®). Viral analyses were performed through the RIDASCREEN® enzyme immunoassay ELISA test kit. The results revealed that the concentration of Giardia spp. in raw sewage varied from 120 to 2,200 cysts/L; this concentration varied from 0.45 to 3.5 cysts/L in treated sewage. The concentration of Cryptosporidium spp. oocysts in raw sewage varied from non-detectable to 28.9 oocysts/L and from non-detectable to 1.05 oocysts/L in treated sewage. The activated sludge process used in ETE promoted a partial removal of parasites such as: Endolimax nana, Entamoeba coli, Entamoeba hystolitica, Ancylostoma sp., Ascaris sp. Fasciola hepatica and Strongyloides stercoralis. Reduction factors varied from 18.2 to 100%. A statistically significant difference was observed between the most probable number of total and thermotolerant coliforms in raw and treated sewage, showing a reduction in the concentration of these indicators after the treatment of sewage in ETE, Ribeirão Preto. No statistically significant correlation was found between the concentrations of total and thermotolerant coliforms in relation to the concentration of Giardia cysts spp. and Cryptosporidium spp. oocysts. Analyzing viruses showed positive responses for Rotavirus and Adenovirus in all the studied samples (raw and treated sewage), with a smaller absorbance in the treated sewage. The results contribute to the identification and evaluation of the prevalence of waterborne microorganisms in treated sewage in ETE, Ribeirão Preto, providing useful data for the definition of public policies for sanitation concerning microbiological quality control in Brazil\'s sewage.

O presente trabalho avaliou o uso e a detecção de óxido de cálcio e ozônio para a inativação de cistos de Giardia spp. e oocistos de Cryptosporidium parvum presentes nos resíduos e na água de lavagem dos filtros gerados após a utilização da tecnologia de ciclo completo com flotação por ar dissolvido (coagulação, floculação, flotação e filtração) em escala de bancada, usando o cloreto de polialumínio PAC como coagulante. Para os ensaios analíticos de recuperação dos protozoários e validação do método foram utilizados as suspensões e o Easyseed® nas matrizes ALF e resíduos. A quantificação dos protozoários foi realizada pelo método de centrifugação direta com a adição de solução de dispersão detergente ICN 7X (MP BIO®) a 1,0% na amostra com a etapa de separação imunomagnética – IMS. As recuperações nos ensaios de qualidade utilizando as suspensões de protozoários foram de 19,86% ± 16,29 e 43,95% ± 11,21, para oocistos de Cryptosporidium e cistos de Giardia respectivamente na matriz ALF, enquanto que para a matriz resíduo foram de 8,16% ± 30,24 para Cryptosporidium e 32,54% ± 46,48 para Giardia. Para os ensaios de recuperação empregando o Easyseed® os valores da matriz ALF foram 2,25% ± 1,37 para Cryptosporidium e 3,75% ± 2,25 para Giardia. No resíduo, a recuperação foi de 4,5% ± 1,50 para Cryptosporidium e 49% ± 1 para Giardia. Para os ensaios com óxido de cálcio na matriz resíduo, a primeira condição com dosagem de 23 mg cal/100mL no tempo de contato de 3 dias a 25° C, não foram encontrados protozoário positivo para o teste IP (iodeto de propídeo), o que deixa esta condição satisfatória. Na segunda condição, com dosagem de 16 mg cal/100mL e tempo de contato de 3 dias a 25°C, foram encontrados protozoários negativos para IP. Para as condições de desinfecção, utilizando ozônio, com tempos de contato 5 min e 10 min e dosagens de 10 mg O3.L-1 e 7,5 mg O3.L-1, respectivamente, poucos organismos foram detectados. Portanto, destaca-se a dificuldade em avaliar a permeabilidade dos protozoários após os ensaios de desinfecção realizados. Faz-se necessário realizar novos ensaios com outras dosagens e tempos de contato. / The present work evaluated the use of calcium oxide and ozone for the inactivation of Giardia spp. and Cryptosporidium parvum oocysts present in the wastes and in the wash water of the filters obtained after the use of the complete cycle technology with dissolved air flotation (coagulation, flocculation, flotation and filtration) on a bench scale using polyaluminium chloride - PAC as a coagulant. For the analytical tests of protozoan recovery and validation of the method, suspensions and Easyseed ® were used in the ALF and residues matrices. Protozoan quantification was performed by the direct centrifugation method with the addition of detergent dispersion solution ICN 7X (MP BIO ®) at 1.0% in the sample with the immunomagnetic separation step - IMS. The recoveries in the quality assays using the protozoal suspensions were 19.86% ± 16.29 and 43.95% ± 11.21 for Cryptosporidium oocysts and Giardia cysts respectively in the ALF matrix, whereas for the residue matrix were 8.16% ± 30.24 for Cryptosporidium and 32.54 ± 46.48 for Giardia. For the recovery assays using Easyseed ® the ALF matrix values were 2.25% ± 1.37 for Cryptosporidium and 3.75% ± 2.25 for Giardia. In the residue, recovery was 4.5% ± 1.50 for Cryptosporidium and 49% ± 1 for Giardia. For the calcium oxide assays in the residue matrix, the first condition with a dosage of 23 mg cal/100mL at the contact time of 3 days at 25°C, no positive protozoan was found for the IP (propidium iodide) test, which leaves is a satisfactory condition. In the second condition with a dosage of 16 mg cal/100mL and contact time of 3 days at 25°C, negative protozoa were found for IP. For the disinfection conditions using ozone with contact times 5 min and 10 min and dosages of 10 mg O3.L-1 and 7.5 mg O3.L-1, respectively, few organisms were detected. Therefore, the difficulty in evaluating the permeability of protozoa after the disinfection tests carried out. Realization of new tests with other dosages and contact times.

No atual cenário de saneamento, há uma escassez de estudos na temática de retenção de microrganismos patogênicos em estações de tratamento de esgotos, onde os principais tratamentos se baseiam na remoção de parâmetros físico-químicos. Uma abordagem mais ampla na remoção destes microrganismos em estações de tratamento e sua quantificação no lodo retido é um passo importante para o aumento do debate acerca da eficiência dos atuais tratamentos utilizados na retenção de patógenos, diminuindo assim a contaminação de corpos d\'água com posteriores problemáticas no âmbito de saúde pública. Desta forma, a proposta do projeto foi avaliar e quantificar a remoção de cistos de Giardia spp., oocistos de Cryptosporidium spp. e Escherichia Coli em sistema de tratamento de esgoto sanitário constituído de tratamento anaeróbio (reator UASB), utilizando dois valores de TDH - 8 horas (Etapa 1) e 12 horas (Etapa 2) - seguido de tratamento em sistema de lodo ativado. O projeto utilizou uma estação de tratamento piloto junto a estação de tratamento da USP São Carlos. Para análises de E. coli e coliformes totais foi utilizada a técnica de pour plate e para detecção de cistos e oocistos de Giardia spp. e Cryptosporidium spp., a técnica de separação imunomagnética (IMS) e técnica de Reação de Imunoflorescência Direta (RID) foi escolhida como método de detecção e quantificação. Para a remoção no reator UASB, a variação de TDH indicou influência na remoção de parâmetros físico-químicos e na retenção de microrganismos, sendo que a utilização de TDH de 12 horas obteve remoções de 0,21 log para cistos Giardia spp. e 0,48 log para oocistos de Cryptosporidium spp. Para o Sistema de Lodo ativado, as remoções foram de 0,48 log e 0,15 para ambos os microrganismos, respectivamente, não havendo influência significativa na retenção com a mudança de TDH do reator UASB. Nas análises em fase sólida, foram observados valores altos de contaminação no lodo, tanto para Giardia spp., quanto para Cryptosporidium spp., onde a viabilidade dos (oo)cistos detectados estiveram na faixa de 30% a 99% nas amostras analisadas. / Currently, the field of sanitation faces a lack of studies on retention of pathogenic microorganisms in wastewater treatment plants, where main treatments are based on the removal of physical and chemical parameters. A broader approach to the removal of pathogens in wastewater treatment systems and their quantification in retained sludge is an important step to increase the debate around the efficiency of prevailing treatments used to retain those microorganisms and to consequently reduce the contamination of water bodies. The present study was conducted to assess and quantify the removal of Giardia spp. cysts, Cryptosporidium spp. oocysts and E. coli in wastewater treatment systems. For the purpose of this research, the treatment consisted of a UASB reactor using two values of hydraulic retention time (HRT) - 8 hours (phase 1) and 12 hours (phase 2) - followed by an activated sludge system. The project was staged in a pilot wastewater treatment plant coupled to USP Sao Carlos\' wastewater treatment system. Pour plate technique was used for analysis of E.coli and total coliforms. IMS was used to detect cysts and oocysts of Giardia spp. and Cryptosporidium spp. DFA (direct immunofluorescence assay) method was chosen to detection and quantification. HRT variation for removal in UASB reactor indicated influence in removal of physical and chemical parameters and in retention of microorganisms. The 12 hours use of HRT obtained removals of 0.21 log for Giardia cysts and 0.48 log for Cryptosporidium oocysts. Activated sludge removals presented 0.48 log and 0.15 log respectively, without significative influence in retention with the HRT\'s change in UASB reactor. High values of contamination were observed during solid-phase analysis in activated sludge, both for Giardia and Cryptosporidium. Here, viability of the (oo)cysts detected was around 30% up to 99% of the analyzed samples.

O propósito do presente estudo foi investigar e monitorar a remoção de cistos de Giardia spp. e oocistos de Cryptosporidium spp. por diferentes processos de uma estação de tratamento de esgoto (ETE) em escala plena, composta basicamente por tratamento preliminar, reator UASB e flotador por ar dissolvido, e verificar a ocorrência desses protozoários no lodo do reator UASB e do flotador. Além disso, avaliou-se a remoção desses parasitos pelo processo de flotação por ar dissolvido em escala de bancada (equipamento Flotateste). Analisou-se a qualidade das amostras a partir de variáveis físicas e químicas, e pela detecção de microrganismos indicadores - E. coli, coliformes totais e Clostridium perfringens. Os métodos de detecção de protozoários se basearam nas etapas de concentração (tripla centrifugação ou filtração em membrana seguida de tripla centrifugação); purificação por separação imunomagnética (IMS); detecção por reação de imunofluorescência direta (RID). As recuperações de cistos variaram de 32,6 a 67,0 % dependendo do método adotado, já para os oocistos as recuperações estiveram na faixa de 5,6 a 12,0 %. Na ETE-Monjolinho foram detectadas significativas quantidades de cistos de Giardia spp. em 100% das amostras de esgoto analisadas, com concentração média de 1,89 x 104 e 2,35 x 102 cistos.L-1 no esgoto bruto e tratado, respectivamente. Já os oocistos de Cryptosporidium spp. foram detectados em 39,0 % das amostras de esgoto, com concentração média de 1,35 x 102 oocistos.L-1 no esgoto bruto e 5,87 oocistos.L-1 em esgoto tratado (após flotador). A remoção global da ETE para remoção de Giardia spp. foi em média 2,03 log. O lodo do reator UASB e lodo do flotador apresentaram altas quantidade de (oo)cistos, constatando-se a tendência desses sistemas em concentrar os (oo)cistos por seus processos físicos. Algumas correlações significativas foram encontradas, como correlação entre a concentração de cistos no lodo e a variável sólidos totais, a concentração de cistos no esgoto bruto e as variáveis cor aparente, DQO total e particulada, e a concentração de cistos no efluente UASB e o microrganismo Clostridium perfringens. Diferentemente do flotador em escala plena, o processo do flotação por ar dissolvido em escala de bancada alcançou elevadas remoções médias de cistos de Giardia spp., entre 2,5 e 2,7 log nas diferentes condições de floculação estudadas. / The aim of this study was to investigate and monitor the removal of Giardia spp. cysts and Cryptosporidium spp. oocysts through different processes of a sewage treatment plant (STP) in full scale, consisted of preliminary treatment, UASB reactor, dissolved air flotator, and to verify the occurrence of the protozoa in the sludge derived from UASB reactor and flotator. Besides that, it was evaluated the removal of these parasites through the dissolved air flotation process in bench scale. It was analyzed the quality of the samples through physical and chemical variables, and detection of indicator microorganisms – E. coli, total coliforms and Clostridium perfringens. The detection methods of protozoa were based on steps of concentration (triple concentration or membrane filtration followed by triple concentration); purification by immunomagnetic separation (IMS); immunofluorescence assay (IFA). The recovery of cysts ranged from 32,6 to 67,0 % according to the adopted method, while the recovery for the oocysts ranged from 5,6 to 12,0 %. It was detected significant quantities of Giardia spp. cysts in 100 % of the analyzed sewage samples at the STP-Monjolinho, with mean concentration of 1,89 x 104 e 2,35 x 102 cysts.L-1in raw sewage and treated sewage, respectively. The oocysts of Cryptosporidium spp. were detected in 39,0 % of the sewage samples, with mean concentration of 1,35 x 102 oocysts.L-1 in raw sewage and 5,87 oocysts.L-1 in treated sewage (after flotator). The overall removal for Giardia spp. was on average of 2,03 log. The sludge from UASB reactor and flotator presented high quantities of (oo)cysts, implying the tendency of these systems to concentrate (oo)cysts through its physical processes. Some correlations were obtained, such as correlation between the concentration of cysts in the sludge and total solids, the concentration of cysts in the raw sewage and apparent color, total and particulate COD (chemical oxygen demand), and the concentration of cysts in the UASB effluent and the microorganism Clostridium perfringens. Unlike the full flotator, the dissolved air flotation in bench scale reached significant mean removal of Giardia spp. cysts, between 2,5 and 2,7 log according to the different flocculation conditions.

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.

O propósito do presente estudo foi investigar e monitorar a remoção de cistos de Giardia spp. e oocistos de Cryptosporidium spp. por diferentes processos de uma estação de tratamento de esgoto (ETE) em escala plena, composta basicamente por tratamento preliminar, reator UASB e flotador por ar dissolvido, e verificar a ocorrência desses protozoários no lodo do reator UASB e do flotador. Além disso, avaliou-se a remoção desses parasitos pelo processo de flotação por ar dissolvido em escala de bancada (equipamento Flotateste). Analisou-se a qualidade das amostras a partir de variáveis físicas e químicas, e pela detecção de microrganismos indicadores - E. coli, coliformes totais e Clostridium perfringens. Os métodos de detecção de protozoários se basearam nas etapas de concentração (tripla centrifugação ou filtração em membrana seguida de tripla centrifugação); purificação por separação imunomagnética (IMS); detecção por reação de imunofluorescência direta (RID). As recuperações de cistos variaram de 32,6 a 67,0 % dependendo do método adotado, já para os oocistos as recuperações estiveram na faixa de 5,6 a 12,0 %. Na ETE-Monjolinho foram detectadas significativas quantidades de cistos de Giardia spp. em 100% das amostras de esgoto analisadas, com concentração média de 1,89 x 104 e 2,35 x 102 cistos.L-1 no esgoto bruto e tratado, respectivamente. Já os oocistos de Cryptosporidium spp. foram detectados em 39,0 % das amostras de esgoto, com concentração média de 1,35 x 102 oocistos.L-1 no esgoto bruto e 5,87 oocistos.L-1 em esgoto tratado (após flotador). A remoção global da ETE para remoção de Giardia spp. foi em média 2,03 log. O lodo do reator UASB e lodo do flotador apresentaram altas quantidade de (oo)cistos, constatando-se a tendência desses sistemas em concentrar os (oo)cistos por seus processos físicos. Algumas correlações significativas foram encontradas, como correlação entre a concentração de cistos no lodo e a variável sólidos totais, a concentração de cistos no esgoto bruto e as variáveis cor aparente, DQO total e particulada, e a concentração de cistos no efluente UASB e o microrganismo Clostridium perfringens. Diferentemente do flotador em escala plena, o processo do flotação por ar dissolvido em escala de bancada alcançou elevadas remoções médias de cistos de Giardia spp., entre 2,5 e 2,7 log nas diferentes condições de floculação estudadas. / The aim of this study was to investigate and monitor the removal of Giardia spp. cysts and Cryptosporidium spp. oocysts through different processes of a sewage treatment plant (STP) in full scale, consisted of preliminary treatment, UASB reactor, dissolved air flotator, and to verify the occurrence of the protozoa in the sludge derived from UASB reactor and flotator. Besides that, it was evaluated the removal of these parasites through the dissolved air flotation process in bench scale. It was analyzed the quality of the samples through physical and chemical variables, and detection of indicator microorganisms – E. coli, total coliforms and Clostridium perfringens. The detection methods of protozoa were based on steps of concentration (triple concentration or membrane filtration followed by triple concentration); purification by immunomagnetic separation (IMS); immunofluorescence assay (IFA). The recovery of cysts ranged from 32,6 to 67,0 % according to the adopted method, while the recovery for the oocysts ranged from 5,6 to 12,0 %. It was detected significant quantities of Giardia spp. cysts in 100 % of the analyzed sewage samples at the STP-Monjolinho, with mean concentration of 1,89 x 104 e 2,35 x 102 cysts.L-1in raw sewage and treated sewage, respectively. The oocysts of Cryptosporidium spp. were detected in 39,0 % of the sewage samples, with mean concentration of 1,35 x 102 oocysts.L-1 in raw sewage and 5,87 oocysts.L-1 in treated sewage (after flotator). The overall removal for Giardia spp. was on average of 2,03 log. The sludge from UASB reactor and flotator presented high quantities of (oo)cysts, implying the tendency of these systems to concentrate (oo)cysts through its physical processes. Some correlations were obtained, such as correlation between the concentration of cysts in the sludge and total solids, the concentration of cysts in the raw sewage and apparent color, total and particulate COD (chemical oxygen demand), and the concentration of cysts in the UASB effluent and the microorganism Clostridium perfringens. Unlike the full flotator, the dissolved air flotation in bench scale reached significant mean removal of Giardia spp. cysts, between 2,5 and 2,7 log according to the different flocculation conditions.

O presente trabalho avaliou o uso e a detecção de óxido de cálcio e ozônio para a inativação de cistos de Giardia spp. e oocistos de Cryptosporidium parvum presentes nos resíduos e na água de lavagem dos filtros gerados após a utilização da tecnologia de ciclo completo com flotação por ar dissolvido (coagulação, floculação, flotação e filtração) em escala de bancada, usando o cloreto de polialumínio PAC como coagulante. Para os ensaios analíticos de recuperação dos protozoários e validação do método foram utilizados as suspensões e o Easyseed® nas matrizes ALF e resíduos. A quantificação dos protozoários foi realizada pelo método de centrifugação direta com a adição de solução de dispersão detergente ICN 7X (MP BIO®) a 1,0% na amostra com a etapa de separação imunomagnética – IMS. As recuperações nos ensaios de qualidade utilizando as suspensões de protozoários foram de 19,86% ± 16,29 e 43,95% ± 11,21, para oocistos de Cryptosporidium e cistos de Giardia respectivamente na matriz ALF, enquanto que para a matriz resíduo foram de 8,16% ± 30,24 para Cryptosporidium e 32,54% ± 46,48 para Giardia. Para os ensaios de recuperação empregando o Easyseed® os valores da matriz ALF foram 2,25% ± 1,37 para Cryptosporidium e 3,75% ± 2,25 para Giardia. No resíduo, a recuperação foi de 4,5% ± 1,50 para Cryptosporidium e 49% ± 1 para Giardia. Para os ensaios com óxido de cálcio na matriz resíduo, a primeira condição com dosagem de 23 mg cal/100mL no tempo de contato de 3 dias a 25° C, não foram encontrados protozoário positivo para o teste IP (iodeto de propídeo), o que deixa esta condição satisfatória. Na segunda condição, com dosagem de 16 mg cal/100mL e tempo de contato de 3 dias a 25°C, foram encontrados protozoários negativos para IP. Para as condições de desinfecção, utilizando ozônio, com tempos de contato 5 min e 10 min e dosagens de 10 mg O3.L-1 e 7,5 mg O3.L-1, respectivamente, poucos organismos foram detectados. Portanto, destaca-se a dificuldade em avaliar a permeabilidade dos protozoários após os ensaios de desinfecção realizados. Faz-se necessário realizar novos ensaios com outras dosagens e tempos de contato. / The present work evaluated the use of calcium oxide and ozone for the inactivation of Giardia spp. and Cryptosporidium parvum oocysts present in the wastes and in the wash water of the filters obtained after the use of the complete cycle technology with dissolved air flotation (coagulation, flocculation, flotation and filtration) on a bench scale using polyaluminium chloride - PAC as a coagulant. For the analytical tests of protozoan recovery and validation of the method, suspensions and Easyseed ® were used in the ALF and residues matrices. Protozoan quantification was performed by the direct centrifugation method with the addition of detergent dispersion solution ICN 7X (MP BIO ®) at 1.0% in the sample with the immunomagnetic separation step - IMS. The recoveries in the quality assays using the protozoal suspensions were 19.86% ± 16.29 and 43.95% ± 11.21 for Cryptosporidium oocysts and Giardia cysts respectively in the ALF matrix, whereas for the residue matrix were 8.16% ± 30.24 for Cryptosporidium and 32.54 ± 46.48 for Giardia. For the recovery assays using Easyseed ® the ALF matrix values were 2.25% ± 1.37 for Cryptosporidium and 3.75% ± 2.25 for Giardia. In the residue, recovery was 4.5% ± 1.50 for Cryptosporidium and 49% ± 1 for Giardia. For the calcium oxide assays in the residue matrix, the first condition with a dosage of 23 mg cal/100mL at the contact time of 3 days at 25°C, no positive protozoan was found for the IP (propidium iodide) test, which leaves is a satisfactory condition. In the second condition with a dosage of 16 mg cal/100mL and contact time of 3 days at 25°C, negative protozoa were found for IP. For the disinfection conditions using ozone with contact times 5 min and 10 min and dosages of 10 mg O3.L-1 and 7.5 mg O3.L-1, respectively, few organisms were detected. Therefore, the difficulty in evaluating the permeability of protozoa after the disinfection tests carried out. Realization of new tests with other dosages and contact times.

No atual cenário de saneamento, há uma escassez de estudos na temática de retenção de microrganismos patogênicos em estações de tratamento de esgotos, onde os principais tratamentos se baseiam na remoção de parâmetros físico-químicos. Uma abordagem mais ampla na remoção destes microrganismos em estações de tratamento e sua quantificação no lodo retido é um passo importante para o aumento do debate acerca da eficiência dos atuais tratamentos utilizados na retenção de patógenos, diminuindo assim a contaminação de corpos d\'água com posteriores problemáticas no âmbito de saúde pública. Desta forma, a proposta do projeto foi avaliar e quantificar a remoção de cistos de Giardia spp., oocistos de Cryptosporidium spp. e Escherichia Coli em sistema de tratamento de esgoto sanitário constituído de tratamento anaeróbio (reator UASB), utilizando dois valores de TDH - 8 horas (Etapa 1) e 12 horas (Etapa 2) - seguido de tratamento em sistema de lodo ativado. O projeto utilizou uma estação de tratamento piloto junto a estação de tratamento da USP São Carlos. Para análises de E. coli e coliformes totais foi utilizada a técnica de pour plate e para detecção de cistos e oocistos de Giardia spp. e Cryptosporidium spp., a técnica de separação imunomagnética (IMS) e técnica de Reação de Imunoflorescência Direta (RID) foi escolhida como método de detecção e quantificação. Para a remoção no reator UASB, a variação de TDH indicou influência na remoção de parâmetros físico-químicos e na retenção de microrganismos, sendo que a utilização de TDH de 12 horas obteve remoções de 0,21 log para cistos Giardia spp. e 0,48 log para oocistos de Cryptosporidium spp. Para o Sistema de Lodo ativado, as remoções foram de 0,48 log e 0,15 para ambos os microrganismos, respectivamente, não havendo influência significativa na retenção com a mudança de TDH do reator UASB. Nas análises em fase sólida, foram observados valores altos de contaminação no lodo, tanto para Giardia spp., quanto para Cryptosporidium spp., onde a viabilidade dos (oo)cistos detectados estiveram na faixa de 30% a 99% nas amostras analisadas. / Currently, the field of sanitation faces a lack of studies on retention of pathogenic microorganisms in wastewater treatment plants, where main treatments are based on the removal of physical and chemical parameters. A broader approach to the removal of pathogens in wastewater treatment systems and their quantification in retained sludge is an important step to increase the debate around the efficiency of prevailing treatments used to retain those microorganisms and to consequently reduce the contamination of water bodies. The present study was conducted to assess and quantify the removal of Giardia spp. cysts, Cryptosporidium spp. oocysts and E. coli in wastewater treatment systems. For the purpose of this research, the treatment consisted of a UASB reactor using two values of hydraulic retention time (HRT) - 8 hours (phase 1) and 12 hours (phase 2) - followed by an activated sludge system. The project was staged in a pilot wastewater treatment plant coupled to USP Sao Carlos\' wastewater treatment system. Pour plate technique was used for analysis of E.coli and total coliforms. IMS was used to detect cysts and oocysts of Giardia spp. and Cryptosporidium spp. DFA (direct immunofluorescence assay) method was chosen to detection and quantification. HRT variation for removal in UASB reactor indicated influence in removal of physical and chemical parameters and in retention of microorganisms. The 12 hours use of HRT obtained removals of 0.21 log for Giardia cysts and 0.48 log for Cryptosporidium oocysts. Activated sludge removals presented 0.48 log and 0.15 log respectively, without significative influence in retention with the HRT\'s change in UASB reactor. High values of contamination were observed during solid-phase analysis in activated sludge, both for Giardia and Cryptosporidium. Here, viability of the (oo)cysts detected was around 30% up to 99% of the analyzed samples.

Surface waters used for irrigation purposes in South Africa have been found to be of poor microbiological quality and to be contaminated with human pathogens. These pathogens can be transferred from contaminated water onto fresh produce and potentially cause human infections. Cryptosporidium and Giardia are waterborne parasitic protozoa that have been found in surface waters worldwide. They can cause morbidity in infected individuals and be lethal when infecting people with compromised immunity. This study was divided into two phases. The first phase was a field survey aimed at determining the incidence of human pathogens Cryptosporidium, Giardia and Salmonella spp. in rivers used for irrigation purposes in South Africa as well as on vegetables irrigated with these rivers. The rivers selected were from three different provinces of South Africa. The relationship between faecal indicators and the presence of Cryptosporidium oocysts and Giardia cysts was also investigated. Out of the 30 water samples analysed, 43% were positive for Cryptosporidium oocysts, 23% tested positive for Giardia cysts and 27% were positive for Salmonella spp. However, no Cryptosporidium oocysts or Giardia cysts were found on the vegetables analysed. No significant differences in the prevalence of Cryptosporidium and Giardia and indicator parameters were observed between the three rivers. Using a logistic regression model, no significant correlations were observed between the incidence of faecal indicators and the presence of Cryptosporidium oocysts and Giardia cysts. In the second phase of the study, the individual and combined effects of chlorine, blanching, blast freezing and microwave heating on Cryptosporidium parvum oocysts inoculated on green peppers were investigated. The viability of the oocysts after treatments was determined using the vital dye propidium iodide. Stained oocysts were counted with a flow cytometer. Chlorine treatments did not significantly affect the viability of the oocysts. Blast freezing affected the viability of 20% of the oocysts. Both microwave heating and blanching affected 93% of oocysts. Combined treatments with chlorine and blast freezing did not affect the viability of the oocysts significantly compared to the control. Combined treatment with chlorine and microwave heating was significantly more effective than microwave heating alone and affected 98.1 % of the oocysts. The data indicate that C. parvum oocysts are sensitive to heat and, to some extent, to freezing temperature but are resistant to chlorine. The results of the survey show the presence of Cryptosporidium and Giardia in irrigation waters and thus a possible health risk associated with the consumption of raw vegetables as those can become contaminated via the irrigation water. The results of the challenge tests indicate that C. parvum oocysts on vegetables are inactivated by blanching and microwave heating but survive blast freezing and exposure to chlorine. Boiling and microwave heating of vegetables should be sufficient to kill C. parvum. On the other hand, ready-to-eat vegetables could be at risk of carrying live C. parvum oocysts as the use of chlorine in washing bath is not expected to inactivate C. parvum oocysts present on vegetables. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Food Science / unrestricted

O lançamento de esgotos domésticos in natura, efluentes das estações de tratamento de esgoto e escoamento superficial, são relatados como importantes causas de poluição das águas superficiais. Sabe-se que a alteração da qualidade das águas dos rios restringe seus múltiplos usos e contribui para o aumento de doenças de veiculação hídrica, em decorrência da exposição oral a bioagentes patogênicos. Neste contexto, o objetivo do presente estudo foi identificar e quantificar bioagentes presentes na água do rio Pardo, Brasil, e estimar o risco de infecção e de doença por Cryptosporidium spp. e Giardia spp. para a população, devido ao uso do rio como fonte de abastecimento público e recreação de contato primário, por meio da abordagem da Avaliação Quantitativa de Risco Microbiológico (AQRM). Durante os anos de 2015 e 2016, foram realizadas seis coletas de amostras da água do rio Pardo (período chuvoso e período seco) em seis pontos, totalizando 36 amostras. Foram realizadas análises de identificação e quantificação de E. coli, Salmonella Não Tifóide, Cryptosporidium spp. e Giardia spp. Para estimativa de risco de infecção e de doença por Cryptosporidium spp. e Giardia spp. (AQRM), foram considerados diferentes populações (crianças e adultos), volumes de água ingerido, concentração de (oo)cistos e duração e frequência da exposição, de acordo com o cenário estabelecido. Os valores médios para E. coli variaram de 6,57 x 101 UFC/100 mL a 6,07 x 103 UFC/100 mL, apresentando diferenças estatisticamente significantes (p < 0,05) entre os períodos chuvoso e seco. As densidades de Salmonella Não Tifóide foram baixas (<0,6473 a 1,55 NMP/100 mL), com frequência de 13,9% das amostras positivas, evidenciando a circulação desse patógeno no ambiente. A concentração de (oo)cistos de Cryptosporidium spp. e Giardia spp. variou de <0,1 a 0,4 oocistos/L e <0,1 a 4,4 cistos/L, respectivamente. Para abordagem da AQRM devido a ingestão da água do rio Pardo usada para abastecimento público, a probabilidade anual de infecção por Cryptosporidium spp. e Giardia spp. foi maior para adultos do que para crianças, sendo que na maioria dos pontos apresentou resultados superiores ao risco anual tolerável pela USEPA (1 x 10-4). No que diz respeito ao uso da água do rio Pardo para recreação de contato primário, a probabilidade diária e anual de infecção, bem como a probabilidade de doenças, foi maior para crianças, seguida de adultos/homens e adultos/mulheres. A probabilidade de criptosporidiose e giardíase esteve abaixo do limite tolerável pela USEPA (3,6 x 10-2), exceto no Ponto 4, em que a estimativa de risco de doença por Giardia spp. para crianças esteve acima deste valor. A presença de bioagentes em amostras de água do rio Pardo pode estar relacionada à poluição das águas por fontes pontuais e difusas. Esses achados refletem a importância de priorizar os recursos para implantação e complementação das Estações de Tratamento de Esgoto na UGRHI 4, a fim de prevenir as doenças de veiculação hídrica em populações que utilizam a água do rio Pardo para abastecimento público e recreação de contato primário / The discharge of domestic sewage, effluents of wastewater treatment plants and surface runoff, are reported as important causes of surface water pollution. It is known that the alteration of river water quality restricts its multiple uses and contributes to the increase of waterborne diseases, due to oral exposure to pathogenic bioagents. In this context, the aim of the present study was to identify and quantify bioagents present in Pardo river water, Brazil, and to estimate the risk of infection and disease by Cryptosporidium spp. and Giardia spp. for the population, due to the use of the river as source of public supply and primary contact recreation, through the approach of Quantitative Microbial Risk Assessment (QMRA). During the years of 2015 and 2016, six samples of water from the Pardo river (rainy and dry season) were collected at six points, totaling 36 samples. Identification and quantification analyzes of E. coli, Non-typhoid Salmonella, Cryptosporidium spp. and Giardia spp. To estimate the risk of infection and disease by Cryptosporidium spp. and Giardia spp. (QMRA), different populations (children and adults), volumes of ingested water, concentration of (oo) cysts, duration and frequency of exposure were considered according to the established scenario. Mean values for E. coli varied from 6.57 x 101 CFU / 100 mL to 6.07 x 103 CFU / 100 mL, showing statistically significant differences (p <0.05) between the rainy and dry season. Non-typhoid Salmonella densities were low (<0.6473 at 1.55 MPN / 100 mL), with a frequency of 13.9% of the positive samples, evidencing the circulation of this pathogen in the environment. Cryptosporidium spp. and Giardia spp. concentration ranged from <0.1 to 0.4 oocysts / L and <0.1 to 4.4 cysts / L, respectively. In order to approach the QMRA due to the ingestion of Pardo river water used for public supply, the probability of annual infection by Cryptosporidium spp. and Giardia spp. was higher for adults than for children, and in most points presented results higher than the risk tolerable by USEPA (1 x 10-4). Regarding the use of Pardo river water for primary contact recreation, the daily and annual probability of infection, as well as the probability of illness, was higher for children, followed by adults / men and adults / women. The probability of cryptosporidiosis and giardiasis was below the limit tolerable by USEPA (3.6 x 10-2), except in Point 4, where the estimated risk of disease by Giardia spp. for children was above this value. The presence of bioagents in Pardo river water may be related to water pollution by point and diffuse sources. These findings reflect the importance of prioritizing the resources for implementation and complementation of wastewater treatment plants at UGRHI 4, in order to prevent waterborne diseases in populations that use Pardo river water for public supply and primary contact recreation