Este trabalho teve o intuito de avaliar a eficiência de remoção de protozoários patogênicos - Giardia spp. e Cryptosporidium spp. - em processos de tratamento de esgoto sanitário por reator UASB, lodos ativados, filtro lento em areia e diferentes desinfetantes. A recuperação de cistos de Giardia e de oocistos de Cryptosporidium, realizada por diferentes metodologias e utilizando ColorSeed®, foi de, respectivamente, 85 e 20% em esgoto bruto, e 62,5 e 17,5% em efluente tratado, quando foi utilizado o método de tripla centrifugação. Cistos de Giardia foram encontrados em 100% as amostras de esgoto pesquisadas, com média de 1,5 x 104 cistos por litro e oocistos de Cryptosporidium em 31,4% com média 3,1 x 10² oocistos por litro, em esgoto bruto. No tratamento biológico por reator UASB seguido de Lodos Ativados, a remoção de cisto de Giardia e esporos de Clostridium perfringens foram estatisticamente menores que as remoções de E. coli e coliformes totais. Não foram encontrados (oo)cistos após o tratamento terciário realizado através da filtração lenta em areia. Houve remoção estatisticamente maior na ETE em escala plena para coliformes totais e Clostridium perfringens. E. coli e cistos de Giardia, em ambas ETEs, apresentaram remoções similares. Elevadas concentrações de (oo)cistos foram encontradas no lodo de esgoto, com grande porcentagem ainda viável. Com relação à desinfecção, entre as bactérias indicadoras, Clostridium perfringens foram mais resistentes ao cloro, ozônio e radiação ultravioleta. O efeito sinérgico, promovido pelas desinfecções sequenciais (clororadiação ultravioleta e ozônio-radiação ultravioleta), foi evidenciado em alguns experimentos para todas as bactérias estudadas. O cloro alterou a fluorescência dos cistos de Giardia e o ozônio, além de alterar a fluorescência, foi capaz de diminuir a concentração de cistos desse microrganismo. Pode-se concluir que as concentrações tanto de microrganismos indicadores como de protozoários patogênicos é bastante elevada, qualquer que seja o tipo de esgoto: bruto, efluente do reator UASB ou efluente do lodos ativados. Isso evidencia o extremo cuidado com que estes efluentes devem ser tratados, para posteriores usos ou lançamento em corpo receptor, em especial devido à presença de (oo)cistos ainda viáveis de Giardia spp. e Cryptosporidium spp. mesmo após o tratamento biológico por lodos ativados, e a necessidade de desinfecção do efluente. / This work aimed to evaluate the efficiency of removal of pathogenic protozoa - Giardia spp. and Cryptosporidium spp. - during wastewater treatment by UASB reactor, activated sludge, slow sand filter and different disinfectants. The recovery of Giardia cysts and of Cryptosporidium oocysts, performed by different methodologies and using ColorSeed®, was respectively of 85 and 20%, in raw wastewater and 62.5 and 17.5% in treated effluent, applying triple centrifugation method. Giardia cysts were found in 100% of the the sewage samples surveyed, with average of 1.5 x 104 cysts per liter and Cryptosporidium oocysts were found in 31.4% with average of 3.1 x 10² oocysts per liter, in raw wastewater. Giardia cyst and Clostridium perfringens spores removals were statistically lower than E. coli and total coliforms removal when applying the biological treatment by UASB reactor followed by Activated Sludge. There were no (oo) cysts after treatment tertiary accomplished by slow sand filtration. There was a statistically higher removal in the full scale WWTP for total coliforms and Clostridium perfringens; however, E. coli and Giardia cysts, in both WWTPs, presented the same removal efficiency. High concentrations of (oo)cysts were found in the sludge sludge, with a high percentage still viable. Regarding disinfection, among the indicating bacteria, Clostridium perfringens was more resistant to chlorine, ozone and ultraviolet radiation. The synergic effect promoted by sequential disinfections (chlorine-ultraviolet radiation and ozone-ultraviolet radiation) was evidenced in some experiments for all the bacteria studied. Chlorine altered the fluorescence of Giardia cysts and ozone, as well as change in fluorescence was able to decrease the concentration of this microorganism. It can be concluded that the concentrations of indicator microorganisms as well as of pathogenic protozoa is very high, regardless the kind of wastewater: raw, UASB reactor effluent or activated sludge effluent. This shows the extreme care that must be taken towards these effluents, for future reuse or simply release in the environment, mainly due to the presence of viable Giardia spp. and Cryptosporidium spp. (oo)cysts even after the activated sludge treatment, and the need of disinfection of the effluent.

Este trabalho teve o intuito de avaliar a eficiência de remoção de protozoários patogênicos - Giardia spp. e Cryptosporidium spp. - em processos de tratamento de esgoto sanitário por reator UASB, lodos ativados, filtro lento em areia e diferentes desinfetantes. A recuperação de cistos de Giardia e de oocistos de Cryptosporidium, realizada por diferentes metodologias e utilizando ColorSeed®, foi de, respectivamente, 85 e 20% em esgoto bruto, e 62,5 e 17,5% em efluente tratado, quando foi utilizado o método de tripla centrifugação. Cistos de Giardia foram encontrados em 100% as amostras de esgoto pesquisadas, com média de 1,5 x 104 cistos por litro e oocistos de Cryptosporidium em 31,4% com média 3,1 x 10² oocistos por litro, em esgoto bruto. No tratamento biológico por reator UASB seguido de Lodos Ativados, a remoção de cisto de Giardia e esporos de Clostridium perfringens foram estatisticamente menores que as remoções de E. coli e coliformes totais. Não foram encontrados (oo)cistos após o tratamento terciário realizado através da filtração lenta em areia. Houve remoção estatisticamente maior na ETE em escala plena para coliformes totais e Clostridium perfringens. E. coli e cistos de Giardia, em ambas ETEs, apresentaram remoções similares. Elevadas concentrações de (oo)cistos foram encontradas no lodo de esgoto, com grande porcentagem ainda viável. Com relação à desinfecção, entre as bactérias indicadoras, Clostridium perfringens foram mais resistentes ao cloro, ozônio e radiação ultravioleta. O efeito sinérgico, promovido pelas desinfecções sequenciais (clororadiação ultravioleta e ozônio-radiação ultravioleta), foi evidenciado em alguns experimentos para todas as bactérias estudadas. O cloro alterou a fluorescência dos cistos de Giardia e o ozônio, além de alterar a fluorescência, foi capaz de diminuir a concentração de cistos desse microrganismo. Pode-se concluir que as concentrações tanto de microrganismos indicadores como de protozoários patogênicos é bastante elevada, qualquer que seja o tipo de esgoto: bruto, efluente do reator UASB ou efluente do lodos ativados. Isso evidencia o extremo cuidado com que estes efluentes devem ser tratados, para posteriores usos ou lançamento em corpo receptor, em especial devido à presença de (oo)cistos ainda viáveis de Giardia spp. e Cryptosporidium spp. mesmo após o tratamento biológico por lodos ativados, e a necessidade de desinfecção do efluente. / This work aimed to evaluate the efficiency of removal of pathogenic protozoa - Giardia spp. and Cryptosporidium spp. - during wastewater treatment by UASB reactor, activated sludge, slow sand filter and different disinfectants. The recovery of Giardia cysts and of Cryptosporidium oocysts, performed by different methodologies and using ColorSeed®, was respectively of 85 and 20%, in raw wastewater and 62.5 and 17.5% in treated effluent, applying triple centrifugation method. Giardia cysts were found in 100% of the the sewage samples surveyed, with average of 1.5 x 104 cysts per liter and Cryptosporidium oocysts were found in 31.4% with average of 3.1 x 10² oocysts per liter, in raw wastewater. Giardia cyst and Clostridium perfringens spores removals were statistically lower than E. coli and total coliforms removal when applying the biological treatment by UASB reactor followed by Activated Sludge. There were no (oo) cysts after treatment tertiary accomplished by slow sand filtration. There was a statistically higher removal in the full scale WWTP for total coliforms and Clostridium perfringens; however, E. coli and Giardia cysts, in both WWTPs, presented the same removal efficiency. High concentrations of (oo)cysts were found in the sludge sludge, with a high percentage still viable. Regarding disinfection, among the indicating bacteria, Clostridium perfringens was more resistant to chlorine, ozone and ultraviolet radiation. The synergic effect promoted by sequential disinfections (chlorine-ultraviolet radiation and ozone-ultraviolet radiation) was evidenced in some experiments for all the bacteria studied. Chlorine altered the fluorescence of Giardia cysts and ozone, as well as change in fluorescence was able to decrease the concentration of this microorganism. It can be concluded that the concentrations of indicator microorganisms as well as of pathogenic protozoa is very high, regardless the kind of wastewater: raw, UASB reactor effluent or activated sludge effluent. This shows the extreme care that must be taken towards these effluents, for future reuse or simply release in the environment, mainly due to the presence of viable Giardia spp. and Cryptosporidium spp. (oo)cysts even after the activated sludge treatment, and the need of disinfection of the effluent.

O objetivo deste trabalho foi avaliar o desempenho da remoção de cistos deGiardia spp. e oocistos de Cryptosporidium parvum, em águas de abastecimento com turbidez elevada, em experimentos em escala de bancada (coagulação, floculação, decantação e filtração). Para tanto, empregou-se o coagulante cloreto de polialumínio – PAC. O método de filtração em membranas foi adotado para a concentração de protozoários, seguido ou não da etapa de purificação por separação imunomagnética – IMS. Os métodos foram avaliados em experimentos de controle de qualidade analítica e o método sem IMS apresentou as seguintes porcentagens de recuperação, 80% ±16,32% para cistos de Giardia spp. e 5% ±10,00% para oocistos de C. parvum. O método com IMS apresentou 31,5%±7,55% de recuperação para cistos de Giardia spp. e 5,75%±3,20% de recuperação para oocistos de C. parvum. Os experimentos demonstraram que não houve melhora na remoção de ambos os protozoários na condição de maior dosagem de coagulante (65 mg.L-1 de PAC e pH 7,29). A condição de menor dosagem de coagulante (25 mg.L-1 de PAC e pH 6,76) apresentou um desempenho melhor, ao contrário de uma expectativa de que a maior dosagem de coagulante pudesse favorecer a remoção destes microrganismos. A condição de menor dosagem apresentou, na água filtrada, 50 e 75% de ausência de identificação de cistos de Giardia e oocistos de C. parvum, respectivamente. A condição de maior dosagem apresentou (oo)cistos na água filtrada de todas amostras analisadas. Estes resultados indicam a importância do controle da coagulação na remoção de protozoários. / The aim of this study was to evaluate the performance of Giardia spp. cysts and Cryptosporidium parvum oocysts removal in a bench scale experiment. The coagulant polyaluminium chloride – PACl was used in this research. The protozoa concentration tests were performed by applying the Membrane Filtration method, with and without Immunomagnetic Separation assay-IMS. The methods were evaluated using control experiments and the method without IMS had the following percentage recovery, 80% ± 16.32% and 5% ±10.00% for Giardia cysts and C. parvum oocysts, respectively. The method with IMS presented 31.5% ± 7.55% and 5.75% ± 3.20% of percentage recovery for Giardia cysts and C. parvum oocysts, respectively. Bench scale experimental results have clearly shown that there was no improvement in protozoa removal using the superior dosage of coagulant. The inferior dosage condition (25 mg.L-1 of PACl and pH 6,76) performed better, which was contrary to what was expected in which a superior dosage of coagulant could favour when removing microorganisms. The inferior dosage condition presented 50% and 75% of absence of Giardia cysts and C. parvum oocysts in the final water, respectively. The second coagulation condition (65 mg.L-1 of PACl and pH 7,29) presented protozoa (oo)cysts in the final water of all the samples examined. These results indicate the importance of coagulation control in protozoa removal.

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

O objetivo deste trabalho foi avaliar o desempenho da remoção de cistos deGiardia spp. e oocistos de Cryptosporidium parvum, em águas de abastecimento com turbidez elevada, em experimentos em escala de bancada (coagulação, floculação, decantação e filtração). Para tanto, empregou-se o coagulante cloreto de polialumínio &#8211; PAC. O método de filtração em membranas foi adotado para a concentração de protozoários, seguido ou não da etapa de purificação por separação imunomagnética &#8211; IMS. Os métodos foram avaliados em experimentos de controle de qualidade analítica e o método sem IMS apresentou as seguintes porcentagens de recuperação, 80% ±16,32% para cistos de Giardia spp. e 5% ±10,00% para oocistos de C. parvum. O método com IMS apresentou 31,5%±7,55% de recuperação para cistos de Giardia spp. e 5,75%±3,20% de recuperação para oocistos de C. parvum. Os experimentos demonstraram que não houve melhora na remoção de ambos os protozoários na condição de maior dosagem de coagulante (65 mg.L-1 de PAC e pH 7,29). A condição de menor dosagem de coagulante (25 mg.L-1 de PAC e pH 6,76) apresentou um desempenho melhor, ao contrário de uma expectativa de que a maior dosagem de coagulante pudesse favorecer a remoção destes microrganismos. A condição de menor dosagem apresentou, na água filtrada, 50 e 75% de ausência de identificação de cistos de Giardia e oocistos de C. parvum, respectivamente. A condição de maior dosagem apresentou (oo)cistos na água filtrada de todas amostras analisadas. Estes resultados indicam a importância do controle da coagulação na remoção de protozoários. / The aim of this study was to evaluate the performance of Giardia spp. cysts and Cryptosporidium parvum oocysts removal in a bench scale experiment. The coagulant polyaluminium chloride &#8211; PACl was used in this research. The protozoa concentration tests were performed by applying the Membrane Filtration method, with and without Immunomagnetic Separation assay-IMS. The methods were evaluated using control experiments and the method without IMS had the following percentage recovery, 80% ± 16.32% and 5% ±10.00% for Giardia cysts and C. parvum oocysts, respectively. The method with IMS presented 31.5% ± 7.55% and 5.75% ± 3.20% of percentage recovery for Giardia cysts and C. parvum oocysts, respectively. Bench scale experimental results have clearly shown that there was no improvement in protozoa removal using the superior dosage of coagulant. The inferior dosage condition (25 mg.L-1 of PACl and pH 6,76) performed better, which was contrary to what was expected in which a superior dosage of coagulant could favour when removing microorganisms. The inferior dosage condition presented 50% and 75% of absence of Giardia cysts and C. parvum oocysts in the final water, respectively. The second coagulation condition (65 mg.L-1 of PACl and pH 7,29) presented protozoa (oo)cysts in the final water of all the samples examined. These results indicate the importance of coagulation control in protozoa removal.

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.

Foodborne illnesses are a significant public health challenge in the United States, with an estimated 9.4 million illnesses annually attributed to the consumption of contaminated food, of which 59% are estimated to be caused by viruses, 39% by bacteria and 2% by parasites. Timely detection and identification of the pathogens causing foodborne outbreaks is vital for the implementation of outbreak control strategies, allowing public health officials to prevent additional illnesses and maintain confidence in the food supply. Public health laboratories employ a variety of traditional and molecular testing techniques to identify foodborne outbreak etiologic agents. One technology is the Luminex XMap® microsphere system, which is also marketed as the Bio-Plex™ 200. This platform has a multiplexing capability with the potential to simultaneously detect up to 100 targets in one reaction. The studies described here show that the combination of two Bio-Plex assays with real-time virus assays and one extraction method provides a flexible foodborne outbreak screening algorithm that potentially identifies an outbreak-associated pathogen on the first day of specimen submission and aids in focusing confirmatory laboratory testing. In these studies, two microsphere-based assays were designed for use on the Bio-Plex 200 system as screening assays for the detection of four enteric protozoa (Giardia intestinalis, Cyclospora cayetanensis, Cryptosporidium parvum, Entamoeba histolytica) and six virulence determinants of shiga toxin-producing Escherichia coli (STEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC) and Shigella spp. Precision and limits of detections were established for both assays. The sensitivity and specificity of the protozoan assay as compared to reference methods ranged from 81.25% to 100% for most targets, while sensitivity for the E. histolytica target was 42.86%. Sensitivity and specificity for the bacterial assay was 100% as compared to reference methods. However, cross-reactivity of the protozoan assay E. histolytica target with E. dispar and of the bacterial assay uidA target with enteropathogenic E. coli strains was noted. Additionally, real-time detection of norovirus and rotavirus nucleic acids extracted with the QIAamp DNA Stool Mini Kit was statistically comparable to detection when extracted with the Ambion® MagMAX™-96 Viral RNA Isolation Kit combined with the KingFisher® Magnetic Particle Processor.

A criptosporidiose é considerada uma das principais infecções por protozoários em aves, e já foi descrita em mais de 30 espécies de aves de várias Ordens, como Anseriformes, Charadriformes, Columbiformes, Galliformes, Passeriformes, Psitaciformes e Struthioniformes. Três espécies de Cryptosporidium infectam aves: Cryptosporidium baileyi, Cryptosporidium galli e Cryptosporidium meleagridis. Além dessas espécies, há vários genótipos distintos geneticamente das espécies de Cryptosporidium já descritas em aves, como os genótipos I, II, III e IV de aves. Há vários relatos de infecção por Cryptosporidium nos tratos gastrintestinal, respiratório e na bursa de Fabricius, resultando em perdas econômicas e mortalidade. O objetivo desse estudo foi a detecção de Cryptosporidium e sua caracterização molecular, em amostras de fezes de aves domésticas e de aves exóticas mantidas em cativeiro no Brasil. Foram coletadas 966 amostras de fezes de 18 famílias de aves. As amostras foram conservadas em solução de dicromato de potássio 2,5% a 4º C, até o processamento. Para purificação e concentração dos oocistos, foi utilizada a técnica de centrífugo-flutuação em solução de Sheather seguida de análise microscópica, em 463 amostras, por meio da técnica de coloração negativa com verde malaquita e de extração do DNA genômico dos oocistos, em amostra positivas à microscopia, ou, alternativamente, a extração de DNA foi realizada, sem a realização prévia de microscopia, em outras 503 amostras. A análise molecular foi realizada por meio da reação de nested-PCR, para amplificação de fragmentos da subunidade 18S do gene do RNA ribossômico e do gene da actina. Foi observada amplificação para Cryptosporidium em 47 (4,86 %) amostras. O sequenciamento dos fragmentos amplificados possibilitou a identificação das três espécies que infectam aves: C. galli> em canário (Serinus canaria), galinha doméstica (Gallus gallus domesticus) e calopsita (Nymphicus hollandicus), C. meleagridis e C. baileyi em galinha doméstica (G. g. domesticus), Cryptosporidium genótipo I de aves em pavão azul (Pavocristatus) e canário (Serinus canaria), Cryptosporidium genótipo III de aves em agapornis (Agapornis roseicolis) e calopsita (N. hollandicus) e Cryptosporidium genótipo II de aves em avestruzes (Struthio camelus). / Cryptosporidiosis is considered a major protozoan infection in birds, and has been described in more than 30 species of birds of various orders, as Anseriformes, Charadriformes, Columbiformes, Galliformes, Passeriformes, Psitaciformes and Struthioniformes. Three species of Cryptosporidium are considered valid in birds: Cryptosporidium baileyi, Cryptosporidium galli and Cryptosporidium meleagridis. Besides these species, there are several genotypes genetically distinct from the species of Cryptosporidium described in birds, as avian genotypes I, II, III and IV. There are several reports of gastrointestinal, respiratory and bursa of Fabricius infections in birds, resulting in major economic losses and mortality. The aim of this study was the detection and molecular characterization of Cryptosporidium spp. in fecal samples of domestic birds and in exotic birds kept in captivity in Brazil. A total of 966 samples from 18 families of birds were collected and stored in 2.5% potassium dichromate solution at 4º C until processing. Oocysts were purified in Sheather sugar solution following microscopic analyses, in 463 samples, by malachite green negative stain and extraction of genomic DNA of oocysts in samples positive by microscopy or, alternatively, DNA extraction was accomplished without previous microscopic analyses in another 503 samples. Molecular analyses were performed using n-PCR for amplification of fragments of the 18S subunit of rRNA gene and of the actin gene. It was observed amplification for Cryptosporidium DNA fragments in 47 (4.86%) samples. Sequencing of amplified fragments and phylogenetic analyses allowed the identification of the three species that infect birds: C. galli in canaries (Serinus canaria), domestic chicken (Gallus gallus domesticus) and calopsita (Nymphicus hollandicus), C. meleagridis and C. baileyi in domestic chicken (G. g. domesticus), Cryptosporidium avian genotype I in peacock (Pavo cristatus) and canary (Serinus canaria), Cryptosporidium avian genotype III in agapornis (Agapornis roseicolis) and cockatiel (N. hollandicus), and Cryptosporidium avian genotype II in ostriches (Struthio camelus).

A criptosporidiose é considerada uma das principais infecções por protozoários em aves, e já foi descrita em mais de 30 espécies de aves de várias Ordens, como Anseriformes, Charadriformes, Columbiformes, Galliformes, Passeriformes, Psitaciformes e Struthioniformes. Três espécies de Cryptosporidium infectam aves: Cryptosporidium baileyi, Cryptosporidium galli e Cryptosporidium meleagridis. Além dessas espécies, há vários genótipos distintos geneticamente das espécies de Cryptosporidium já descritas em aves, como os genótipos I, II, III e IV de aves. Há vários relatos de infecção por Cryptosporidium nos tratos gastrintestinal, respiratório e na bursa de Fabricius, resultando em perdas econômicas e mortalidade. O objetivo desse estudo foi a detecção de Cryptosporidium e sua caracterização molecular, em amostras de fezes de aves domésticas e de aves exóticas mantidas em cativeiro no Brasil. Foram coletadas 966 amostras de fezes de 18 famílias de aves. As amostras foram conservadas em solução de dicromato de potássio 2,5% a 4º C, até o processamento. Para purificação e concentração dos oocistos, foi utilizada a técnica de centrífugo-flutuação em solução de Sheather seguida de análise microscópica, em 463 amostras, por meio da técnica de coloração negativa com verde malaquita e de extração do DNA genômico dos oocistos, em amostra positivas à microscopia, ou, alternativamente, a extração de DNA foi realizada, sem a realização prévia de microscopia, em outras 503 amostras. A análise molecular foi realizada por meio da reação de nested-PCR, para amplificação de fragmentos da subunidade 18S do gene do RNA ribossômico e do gene da actina. Foi observada amplificação para Cryptosporidium em 47 (4,86 %) amostras. O sequenciamento dos fragmentos amplificados possibilitou a identificação das três espécies que infectam aves: C. galli> em canário (Serinus canaria), galinha doméstica (Gallus gallus domesticus) e calopsita (Nymphicus hollandicus), C. meleagridis e C. baileyi em galinha doméstica (G. g. domesticus), Cryptosporidium genótipo I de aves em pavão azul (Pavocristatus) e canário (Serinus canaria), Cryptosporidium genótipo III de aves em agapornis (Agapornis roseicolis) e calopsita (N. hollandicus) e Cryptosporidium genótipo II de aves em avestruzes (Struthio camelus). / Cryptosporidiosis is considered a major protozoan infection in birds, and has been described in more than 30 species of birds of various orders, as Anseriformes, Charadriformes, Columbiformes, Galliformes, Passeriformes, Psitaciformes and Struthioniformes. Three species of Cryptosporidium are considered valid in birds: Cryptosporidium baileyi, Cryptosporidium galli and Cryptosporidium meleagridis. Besides these species, there are several genotypes genetically distinct from the species of Cryptosporidium described in birds, as avian genotypes I, II, III and IV. There are several reports of gastrointestinal, respiratory and bursa of Fabricius infections in birds, resulting in major economic losses and mortality. The aim of this study was the detection and molecular characterization of Cryptosporidium spp. in fecal samples of domestic birds and in exotic birds kept in captivity in Brazil. A total of 966 samples from 18 families of birds were collected and stored in 2.5% potassium dichromate solution at 4º C until processing. Oocysts were purified in Sheather sugar solution following microscopic analyses, in 463 samples, by malachite green negative stain and extraction of genomic DNA of oocysts in samples positive by microscopy or, alternatively, DNA extraction was accomplished without previous microscopic analyses in another 503 samples. Molecular analyses were performed using n-PCR for amplification of fragments of the 18S subunit of rRNA gene and of the actin gene. It was observed amplification for Cryptosporidium DNA fragments in 47 (4.86%) samples. Sequencing of amplified fragments and phylogenetic analyses allowed the identification of the three species that infect birds: C. galli in canaries (Serinus canaria), domestic chicken (Gallus gallus domesticus) and calopsita (Nymphicus hollandicus), C. meleagridis and C. baileyi in domestic chicken (G. g. domesticus), Cryptosporidium avian genotype I in peacock (Pavo cristatus) and canary (Serinus canaria), Cryptosporidium avian genotype III in agapornis (Agapornis roseicolis) and cockatiel (N. hollandicus), and Cryptosporidium avian genotype II in ostriches (Struthio camelus).

PhD (Microbiology) / Department of Microbiology / Background: Diarrheal diseases have a major effect on human health, Globally; it is second only to pneumonia as a leading cause of death among children under five. They are due to a variety of infectious and non-infectious agents; including Entamoeba spp. Entamoeba histolytica is an invasive enteric protozoan parasite that causes amebiasis. Amebiasis is frequent in communities without clean water and poor sanitation, which include low-income South African populations in Giyani and Pretoria. In these populations, the amount of diarrhea caused by Entamoeba histolytica inclusive of all ages, sexes and HIV status is uncertain. Diagnosis of the parasite is usually by microscopy. However, microscopy lacks sensitivity and specificity, therefore it is not reliable. Fortunately, molecular diagnostic tests have been developed to detect different Entamoeba species in humans. It is known that the parasite E. histolytica causes asymptomatic and symptomatic diseases. However, the transition from colonization to disease is still unclear. While parasite and host factors, as well as environmental conditions influence the infection outcome, there is currently no clear explanation of wide variation in the presentation of the disease. This could suggest that there are other factors affecting the disease outcome. A better understanding of these factors as well as their role in disease remains target objectives of modern scientists and it will definitely help in the fight against the disease. In spite of the emerging evidence that the host microbiome, parasite burden and the inflammatory response contribute to the virulence of E. histolytica, their roles have never been defined in developing regions such as Giyani and Pretoria. In addition, the present study hypothesized that co-infections with E. histolytica and secretion of extracellular vesicles/exosomes have a significant impact on the virulence of E. histolytica. Little has been explored or elucidated about responses triggered by other enteropathogens/ameba interplay that could be important in the induction of tissue invasion and disease and also how E. histolytica/enteropathogens interplay in these infections has not been determined. Therefore, the knowledge of this interplay could help in understanding how this modifies disease manifestations by modulating pathogen virulence and the host response. The use of secretion systems is an essential biological process exploited by pathogenic microorganisms to promote survival and spread of the pathogen, which in turn exacerbate the infection. The study of extracellular vesicles (EVs) released by pathogens is a new and exciting field that may realistically contribute to a better understanding of the pathogenic process of E. histolytica and provide alternate control strategies. Aim and objective of the study: The overall aim of the study was to determine the impact of enteric pathogens and secreted extracellular vesicles on amebic virulence and the outcome of infection. This aim was addressed in through a series of six primary objectives, which were: a. To investigate the distribution and prevalence of protozoan parasites in South Africa. b. To investigate novel species of Entamoeba circulating in the South African population. ix c. To elucidate the impact of gut microbiota and immune response during amebic infection. d. To determine the role of Entamoeba histolytica macrophage inhibitory factor (EhMIF) during amebic infection. e. To investigate the impact of co-infections on the outcome of amebiasis. f. To determine the presence of secreted extracellular vesicles/exosomes in Entamoeba histolytica. Brief methodology and results: A modified and validated Taqman qPCR assay (with taqman probes and genus specific primers) was used for amplification and target detection. This assay was used to investigate the distribution and prevalence of protozoan parasites (Cryptosporidium spp and Giardia lamblia) in South Africa, the assay was considered superior for this project because it is more sensitive than conventional PCR and it can be used to detect multiple infection targets. This assay allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes. A total of 484 stool samples collected from diarrheal and non-diarrheal patients from rural and urban communities of South Africa were studied. The overall prevalence of parasites (Giardia lamblia and Cryptosporidium spp) in rural and urban patients were found to be 49% (112/227) and 21% (54/257) respectively (p= < 0.0001). The distribution of specific pathogens in rural areas was Cryptosporidium spp (20%) and Giardia lamblia (14%). Our findings showed no significant difference in parasitic infections between gender and the age of the participants (Chapter 3). The discovery of novel species is of great importance to human health. We have recently discovered stools positive for Entamoeba organisms by microscopy but PCR negative for known Entamoeba species. This led to the hypothesis that novel species of Entamoeba are present in the South African population. A comprehensive assay was used which included probes to identify Entamoeba bangladeshi from diarrheal and non-diarrheal participants. A sensitive qPCR assays and amplicon sequencing was used to detect Entamoeba spp, Prevotella copri and Enterobacteriaceae. Interestingly, E. bangladeshi was identified in the South African population. Entamoeba was present in 27% (E. histolytica 8.5% (41/484), E. dispar 8% (38/484), and E. bangladeshi 4.75% (23/484) E. moshkovskii was not detected in the present study. We were also able to observe changes in the host microbiome and the parasite burden associated with E. histolytica infections in S. African diarrhea cases versus asymptomatic controls but not with E. bangladeshi or E. dispar. In E. histolytica positive samples the level of both parasite and P. copri were lower in non-diarrheal samples (p=0.0034) (Chapter 4). There is accumulating evidence that the inflammatory response contributes to injury. Little is known about the key parasite mediators of host mucosal immunopathology. This study hypothesized that migration inhibitory factor (MIF) mediates the destructive host inflammatory response seen in amebic colitis. To determine the role of EhMIF during amebic infection, we used a genetic approach to test the effect of EhMIF on mucosal inflammation. We found that EhMIF induces IL-8 secretion from intestinal epithelial cells. Mice treated with antibodies that specifically block EhMIF had reduced chemokine expression and neutrophil infiltration in the mucosa. In addition to antibody-mediated neutralization, mice infected with parasites overexpressing EhMIF had increased chemokine expression, neutrophil influx and mucosal damage. We also found that the concentration of EhMIF correlated with the level of intestinal inflammation in persons with intestinal amebiasis. Together, our results reveal a novel parasite mediator of mucosal inflammation and support MIF homologs as potential immunomodulatory targets (Chapter 5). To investigate the impact of co-infections on the outcome of amebiasis, we analyzed the co-occurence of E. histolytica with other enteropathogens known to cause diarrheal infections, such as Shigella/EIEC (IpaH), Campylobacter (cadf), Enterotoxigenic E. coli (STh), Norovirus GII and Adenovirus (Hexon). The results were compared with those obtained with E. histolytica that were not interacted with enteropathogens and with E. histolytica interacted with enteropathogens. The impact of multiple infections on the outcome of the infection was compared between nondiarrheal and diarrheal stool samples. It was found that co-infections with two pathogens were associated with diarrhea compared to single infections. Moreover, Norovirus GII, Campylobacter (Cadf) and co-infections were associated with diarrhea in the study population. This study did not show any significant impact of pathogens co-infecting with E. histolytica on the outcome of amebic infection (Chapter 6). The presence of secreted extracellular vesicles/Exosomes in Entamoeba histolytica was determined by using the Pathogenic ameba strains (HM-1:IMSS or HM-1:IMSS (Sub-strain-US) from petri’s lab to purify exosomes using the commercially available kit to isolate exosomes (total exosomes isolation kit). Our study for the first time revealed that E. histolytica does secrete Evs. This finding increases the appreciation that all organisms are likely to secrete these EVs (Chapter 7). However, the impact of these EVs on the pathogenesis of E. histolytica needs further investigations. Conclusion: This study has contributed significantly to our knowledge on infectious diarrhea and the diversity of Entamoeba species by providing new data on the rate and prevalence of Entamoeba diarrheal infections and their distribution in the South African population. Our study describes for the first time the presence of E. bangladeshi in the South African population. Furthermore, our results reveal a novel parasite mediator of mucosal inflammation and support MIF homologs as potential immunomodulatory targets. This study also, for the first time revealed that E. histolytica does secrete EVs. The results from this work will undoubtedly open an exciting research to establish a deeper understanding of the function and role of these vesicles in amebic infection. We encourage public health interventions like health education programs and improvement of sanitation and hygiene in these populations. Molecular diagnostics should be used for specific diagnostic in clinical settings. / NRF