Molecular characterization of waterborne protozoa in surface water and sediment in Brazil: a taxonomic survey of ciliated protozoa and their correlation with Giardia duodenalis and Cryptosporidium spp.

The detection of Giardia duodenalis and Cryptosporidium spp. was performed, along with the identification of the ciliated protozoa biodiversity, to evaluate the correlation between these protozoa in freshwater quality monitoring. Water and sediment samples from two sites in the Atibaia River (Campinas, São Paulo, Brazil) were collected monthly for 2 years (n = 96). Pathogenic protozoa in water and sediment were detected by using immunomagnetic separation, followed by visualization by immunofluorescence assay (IFA). All positive aliquots in IFA were subjected to DNA extraction and subsequently nested PCR. Qualitative (in vivo observation and silver impregnation) and quantitative (in vivo enumeration) analyses were performed for the ciliated protozoa. Giardia cysts were detected in 62.5% of the surface water samples and Cryptosporidium spp. in 25.0%. In the sediment, cysts were detected in 35.4% samples and oocysts in 16.6%. A total of 57 samples positive for Giardia cysts were subjected to sequencing, 40 of which were harboring G. duodenalis (24 were characterized as sub-assemblage AII). For ciliated protozoa, 73 taxa belonging to 53 genera were identified over the period of the study. These results revealed a high degree of contamination by waterborne protozoa in the main water source which supplies drinking water for more than one million people in Campinas (São Paulo), highlighting the need for continuous monitoring of this catchment site. In addition, the present study provides important data regarding the sources of the water body degradation, i.e., fecal contamination of human origin, in addition to the survey of the ciliated protozoa.

Occurrence and diversity of ciliated protozoa in the most commonly consumed leafy greens in Brazil: a qualitative study

Ciliated protozoa are an ubiquitous group of eukaryotes that have been poorly reported on leafy greens. The present study aimed to verify the occurrence and diversity of ciliated protozoa in the leaves and roots of three of the most commonly consumed leafy greens in Brazil ­ lettuce, rocket and coriander. The vegetable samples were washed by manual agitation (3 minutes) in two different media (mineral water and Page ́s Amoeba Saline solution). After washing, the contents were incubated in Petri dishes and aliquots were removed for microscopic identification and in vivo observation. A total of 21 ciliated protozoa species were found, most of which were bacterivorous. Leafy greens have commonly been associated with foodborne outbreaks and ciliated protozoa, which although they are not a Public Health concern, can act as "Trojan Horses" harboring bacteria, viruses and other protozoa cysts and oocysts and can suggest a new route towards microbiological quality related to the food chain. This is the first report of ciliated protozoa on leafy greens consumed in Brazil. (AU)

Os protozoários ciliados constituem um grupo onipresente de eucariotos pouco relatados em vegetais folhosos. O presente estudo teve como objetivo verificar a ocorrência e a diversidade de protozoários ciliados nas folhas e raízes de três dos vegetais folhosos mais consumidos no Brasil - alface, rúcula e coentro. As amostras de vegetais foram lavadas por agitação manual (3 minutos) em dois meios diferentes (água mineral e Solução Salina para Ameba). Após a lavagem, o conteúdo foi incubado em placas de Petri e alíquotas foram retiradas para identificação microscópica e observação in vivo dos organismos. Um total de 21 espécies de protozoários ciliados foi encontrado, a maioria das quais era bacterívora. Tais hortaliças têm sido comumente associadas a surtos transmitidos por alimentos e os protozoários ciliados que, embora não sejam um problema de saúde pública, podem atuar como "Cavalos de Tróia" ao abrigar bactérias, vírus além de cistos e oocistos de protozoários o que pode sugerir uma nova rota para a avaliação da qualidade microbiológica relacionada à cadeia alimentar. Este é o primeiro relato de protozoários ciliados em folhas verdes consumidas no Brasil. (AU)

Occurrence and diversity of ciliated protozoa in the most commonly consumed leafy greens in Brazil: a qualitative study / Ocorrência e diversidade de protozoários ciliados nos vegetais folhosos mais consumidos no Brasil: um estudo qualitativo

Ciliated protozoa are an ubiquitous group of eukaryotes that have been poorly reported on leafy greens. The present study aimed to verify the occurrence and diversity of ciliated protozoa in the leaves and roots of three of the most commonly consumed leafy greens in Brazil – lettuce, rocket and coriander. The vegetable samples were washed by manual agitation (3 minutes) in two different media (mineral water and Page ́s Amoeba Saline solution). After washing, the contents were incubated in Petri dishes and aliquots were removed for microscopic identification and in vivo observation. A total of 21 ciliated protozoa species were found, most of which were bacterivorous. Leafy greens have commonly been associated with foodborne outbreaks and ciliated protozoa, which although they are not a Public Health concern, can act as “Trojan Horses” harboring bacteria, viruses and other protozoa cysts and oocysts and can suggest a new route towards microbiological quality related to the food chain. This is the first report of ciliated protozoa on leafy greens consumed in Brazil.

Os protozoários ciliados constituem um grupo onipresente de eucariotos pouco relatados em vegetais folhosos. O presente estudo teve como objetivo verificar a ocorrência e a diversidade de protozoários ciliados nas folhas e raízes de três dos vegetais folhosos mais consumidos no Brasil - alface, rúcula e coentro. As amostras de vegetais foram lavadas por agitação manual (3 minutos) em dois meios diferentes (água mineral e Solução Salina para Ameba). Após a lavagem, o conteúdo foi incubado em placas de Petri e alíquotas foram retiradas para identificação microscópica e observação in vivo dos organismos. Um total de 21 espécies de protozoários ciliados foi encontrado, a maioria das quais era bacterívora. Tais hortaliças têm sido comumente associadas a surtos transmitidos por alimentos e os protozoários ciliados que, embora não sejam um problema de saúde pública, podem atuar como “Cavalos de Tróia” ao abrigar bactérias, vírus além de cistos e oocistos de protozoários o que pode sugerir uma nova rota para a avaliação da qualidade microbiológica relacionada à cadeia alimentar. Este é o primeiro relato de protozoários ciliados em folhas verdes consumidas no Brasil.

Giardia spp. and Cryptosporidium spp. removal efficiency of a combined fixed-film system treating domestic wastewater receiving hospital effluent.

Giardia and Cryptosporidium have caused numerous outbreaks of diarrhea as a result of the ingestion of water contaminated with sewage. In Brazil, the efficiency of Giardia and Cryptosporidium removal by combined fixed-film systems has rarely been studied. The aims of the present study were therefore to verify the removal efficiency of Giardia and Cryptosporidium by a combined system (anaerobic/anoxic filter and aerated submerged biofilter) and to perform the genetic characterization of these parasites. The (oo)cysts were detected by centrifuge concentration and membrane filtration from raw sewage, effluents, adhered biomass, and sludge samples. Immunofluorescence assay and differential interference contrast microscopy were used for the visualization of the (oo)cysts. Nested PCR was applied to confirm Giardia and Cryptosporidium. Giardia and Cryptosporidium were detected in 27% and 5.5% of the 144 analyzed samples of raw sewage and effluents, respectively. A total of 33,000 cysts/L were recovered in the adhered biomass samples (n = 25) from different points of the aerated submerged biofilter, while 6000 oocysts/L were registered in a single point. An average of 11,800 cysts/L were found in the sludge samples (n = 5). The combined system exhibited a removal efficiency of Giardia cysts of 1.8 ± 1.0 log removal. The C and BIV assemblages of Giardia were identified in the raw sewage while AII was found in the treated effluent sample. It was not possible to calculate the removal efficiency of Cryptosporidium oocysts by the combined system. The combined system exhibited some potential as a suitable treatment for the removal of parasites from sewage.

Enterocytozoon bieneusi detected by molecular methods in raw sewage and treated effluent from a combined system in Brazil.

BACKGROUND: Enterocytozoon bieneusi are the most common microsporidia associated with different clinical manifestations such as diarrhoea, respiratory tract inflammation and acalculous cholecystitis, especially in immunocompromised patients. Infection usually occurs by ingestion of food and water contaminated with spores, but can also result from direct contact with spores through broken skin, eye lesions, and sexual transmission, depending on the microsporidian species. Although there are reports of E. bieneusi found in humans and animals in Brazil, there are no published studies of environmental samples examined by molecular methods. OBJECTIVES: The purpose of this study was to verify the presence of E. bieneusi in raw sewage and treated effluent from a combined system by molecular methods. METHODS: Raw sewage and treated effluent samples collected from a combined system were analysed for the presence of E. bieneusi using the internal transcriber spacer (ITS) region of E. bieneusi by nested polymerase chain reaction. FINDINGS: The analysis revealed E. bieneusi presence and a novel genotype (EbRB) in one raw sewage sample and one treated effluent. MAIN CONCLUSIONS: The presence of E. bieneusi in final effluent indicates that the combined system may not remove microsporidian spores. This study is the first report of E. bieneusi in environmental samples in Brazil.

Enterocytozoon bieneusi detected by molecular methods in raw sewage and treated effluent from a combined system in Brazil

BACKGROUND Enterocytozoon bieneusi are the most common microsporidia associated with different clinical manifestations such as diarrhoea, respiratory tract inflammation and acalculous cholecystitis, especially in immunocompromised patients. Infection usually occurs by ingestion of food and water contaminated with spores, but can also result from direct contact with spores through broken skin, eye lesions, and sexual transmission, depending on the microsporidian species. Although there are reports of E. bieneusi found in humans and animals in Brazil, there are no published studies of environmental samples examined by molecular methods. OBJECTIVES The purpose of this study was to verify the presence of E. bieneusi in raw sewage and treated effluent from a combined system by molecular methods. METHODS Raw sewage and treated effluent samples collected from a combined system were analysed for the presence of E. bieneusi using the internal transcriber spacer (ITS) region of E. bieneusi by nested polymerase chain reaction. FINDINGS The analysis revealed E. bieneusi presence and a novel genotype (EbRB) in one raw sewage sample and one treated effluent. MAIN CONCLUSIONS The presence of E. bieneusi in final effluent indicates that the combined system may not remove microsporidian spores. This study is the first report of E. bieneusi in environmental samples in Brazil.

Ciliated protozoa community of a combined UASB-activated sludge system in southeastern Brazil.

The aims of the present study were (1) to evaluate the abundance and taxonomic composition of ciliated protozoa in the activated sludge of a full-scale combined anaerobic-aerobic system operating in a tropical country and (2) to study the relationship between the effluent quality, the physicochemical variables, and the ciliates present in the operating system. The total ciliate fauna of the activated sludge of the Piçarrão Wastewater Treatment Plant (Piçarrão WWTP) was composed of 36 morphospecies belonging to 33 genera. These included 21 species observed in the activated sludge samples on the day of collection and 15 species found in cultures. The activated sludge of the Piçarrão WWTP contained a diversified ciliate community composed mainly of indicator organisms. The most frequently occurring morphospecies were Aspidisca cicada, Vorticella spp., Gastronauta aloisi, Acineria uncinata, and Epistylis plicatilis complex. These results showed that satisfactory operating conditions prevailed at the Piçarrão WWTP. In the combined UASB-activated sludge system, the presence of Aspidisca cicada suggests the occurrence of denitrification in the process while the presence of Acineria uncinata and G. alosi indicates the removal of carbonaceous organic matter.

First report of predation of Giardia sp. cysts by ciliated protozoa and confirmation of predation of Cryptosporidium spp. oocysts by ciliate species.

Ciliated protozoa are important components of the microbial food web in various habitats, especially aquatic environments. These organisms are useful bioindicators for both environmental quality assessment and the wastewater purification process. The pathogenic parasitic protozoan species Giardia and Cryptosporidium represent a significant concern for human health, being responsible for numerous disease outbreaks worldwide. The predation of cysts and oocysts in 15 ciliate species from water and sewage samples collected in Campinas, São Paulo, Brazil were verified under laboratory conditions. The ciliated protozoan species were selected based on their mode of nutrition, and only bacterivorous and suspension-feeders were considered for the experiments. The species Blepharisma sinuosum, Euplotes aediculatus, Sterkiella cavicola, Oxytricha granulifera, Vorticella infusionum, Spirostomum minus, and Stentor coeruleus ingested cysts and oocysts, the resistance forms of Giardia spp. and Cryptosporidium spp., respectively. This is the first time that the ingestion of Giardia cysts by ciliated protozoa has been reported. These findings may contribute to a better understanding of the biological removal of these pathogens from aquatic environments.

Elaboración de dosímetros de alanina y sus posibles aplicaciones en riesgos profesionales / Elaboração de dosímetros de alanina e suas possíveis aplicações em riscos profissionais / Elaboration of alanine dosimeters and their possible applications in professional risks

Desarrollar dosímetros de alanina y alanina/parafina con miras a atender la creciente utilización de la radiación ionizante a nivel tecnológico. Materiales y métodos. Los radicales libres producidos por la radiación ionizante en la alanina se detectan por resonancia magnética electrónica (ESR); la parafina se utiliza como aglutinante. La metodología de preparación incluye: elaboración, irradiación, lectura y análisis de datos para los dos tipos de dosímetros. Los espectros ESR estudiados corresponden a dosímetros de alanina con masas entre 60 y 120 mg e irradiados con dosis de 10 y 20 Gy, y dosímetros de alanina/parafina de 160 mg e irradiados con diferentes dosis en un haz de fotones de 4 MV. Resultados. La intensidad de los espectros (las cinco líneas características) depende de las dosis recibidas por los dosímetros y su relación es lineal. Con alanina pura y una dosis de 10 Gy la mínima cantidad requerida fue 120 mg; la compactación del cilindro lograda no fue suficiente para evitar la fragmentación parcial del dosímetro. La parafina no tiene señal paramagnética; en los dosímetros de alanina/parafina (cilindros de longitud 13 mm y diámetro 3.5 mm, en la relación 80:20, y buena dureza) se estudió la respuesta señal ESR-dosis en un rango entre 20 y 120 Gy. Conclusiones. Los cilindros de alanina/parafina fabricados tienen una dureza apropiada para ser manipulados como dosímetros de radiación ionizante; su reproducibilidad y la efectividad en la acumulación de dosis es buena...

Elaboration of alanine dosimeters and their possible applications in professional risks. Objective. To develop dosimeters of alanine and alanine/paraffin in order to meet the growing use of ionizing radiation at the technological level. Materials and methods. Free radicals produced by ionizing radiation in alanine are detected by electron spin resonance (ESR); paraffin is used as binder. The methodology ofpreparation includes: elaboration, irradiation, data collection and data analysis for the two types of dosimeters. The ESR spectra studied correspond to alanine dosimeters with masses between 60 and 120 mg and irradiated with doses of 10 and 20 Gy, and alanine/paraffin dosimeters with 160 mg and irradiated with different doses in a beam of photons 4 MV. Results. The intensity of the spectra (thecharacteristic five lines) depends on the doses received by the dosimeters and their relationship is linear. With pure alanine and a dose of 10 Gy, the minimum amount required was 120 mg; the achieved cylinder compaction was not sufficient to avoid the partial fragmentation of the dosimeter. Paraffin has no paramagnetic signal; in the alanine/paraffin dosimeters (cylinders of 13 mm in length and 3.5 mm of diameter, with a ratio 80:20, and good hardness) the response signal ESR-dose was studied in a range between 20 and 120 Gy. Conclusions. The cylinders of alanine/paraffin manufactured have an appropriate hardness to be handled as dosimeters of ionizing radiation; their reproducibility and effectiveness in the accumulation of dose is good...

Elaboração de dosímetros de alanina e suas possíveis aplicações em riscos profissionais. Objetivo. Desenvolver dosímetros de alanina e alanina/parafina com a finalidade de satisfazer a crescente utilização de radiações ionizantes ao nível tecnológico. Materiais e métodos. Os radicais livres produzidos pela radiação ionizante na alanina são detectados por ressonância magnética eletrônica (EPR); a parafina é utilizada como aglutinante. O método de preparação inclui: elaboração, irradiação, leitura e analise dos dados para os dois tiposde dosímetros. Os espectros ESR estudados correspondem a dosímetros de alanina com massas entre 60 e 120 mg e irradiados com doses de 10 e 20 Gy, e dosímetros de alanina/parafina de 160 mg e irradiados com diferentes doses de um feixe de fótons de 4 MV. Resultados.A intensidade dos espectros (as cinco linhas características) depende das doses recebidas pelos dosímetros e sua relação é linear. Com lanina pura e uma dose de 10 Gy a mínima quantidade requerida foi de 120 mg; a compactação do cilindro alcançada não foi suficiente para evitar a fragmentação parcial do dosímetro. A parafina não tem sinal paramagnético; nos dosímetros de alanina/parafina (cilindros de 13 mmde comprimento e 3,5 mm de diâmetro, na proporção 80:20 e boa dureza) foi estudada a resposta a sinal ESR-dose em um intervalo entre 20 e 120 Gy. Conclusões. Os cilindros de alanina/parafina fabricados têm uma dureza adequada para ser tratados como dosímetros de radiação ionizante; sua reprodutibilidade e eficiência no acúmulo de dose é bom...