Memoria para optar al Título Profesional de Médico Veterinario / En el presente estudio se analizaron 153 muestras de heces frescas de gatos domésticos sanos, habitantes de la ciudad de Santiago, Chile, en busca del protozoario zoonótico, Cryptosporidium spp. Las muestras fueron recolectadas en el periodo abril 2009- septiembre 2010, y luego procesadas en el Laboratorio de Parasitología de la Facultad de Ciencias Veterinarias y Pecuarias de la Universidad de Chile, mediante tinción de Ziehl-Neelsen y posterior observación con microscopía óptica, obteniéndose una muestra positiva (0,65%), y cinco sospechosas (3,3%). La muestra positiva pertenecía a una hembra, de 6 meses de edad (grupo etario Kitten), outdoor, de la comuna El Bosque

O protozoário parasita Cryptosporidium emergiu como um dos mais importantes contaminantes da água responsável por vários surtos de criptosporidiose, afetando mais de 427.000 indivíduos em todo o mundo. Até hoje, pelo menos oito espécies do gênero Cryptosporidium foram descritas, mas somente o C. parvum têm sido associado às doenças gastroentestinais em humanos. A criptosporidiose pode ser fatal para imunocomprometidos e pode debilitar severamente indivíduos imunocompetentes. Os oocistos de Cryptosporidium são resistentes às pressões ambientais, podendo sobreviver por vários meses no ambiente aquático e são também resistentes à desinfecção por cloro utilizada no tratamento convencional de água. Este estudo teve como objetivos determinar a ocorrência e densidade de Cryptosporidium em amostras de água superficiais e tratadas (após floculação, coagulação, sedimentação, filtração e desinfecção) coletadas em duas Estações de Tratamento de Água da cidade de São Paulo. A relação entre os parâmetros da qualidade da água e a ocorrência de oocistos de Cryptosporidium também foi analisada. As amostras de água foram coletadas em intervalos mensais durante o período de um ano. Estas amostras foram concentradas por ''precipitação do carbonato de cálcio'' (VESEY et alii, 1993a) e através da técnica da ''membrana filtrante'' (ALDOM & CHAGLA, 1994). Os oocistos foram identificados pela técnica de imunofluorescência direta e a presença destes foi confirmada por microscopia de contraste de fase. Os níveis de coliformes totais e E.coli nas amostras de água foram determinados pela técnica dos tubos múltiplos, empregando substrato fluorogênico e cromogênico (Colilert 18, Iddex). De um total de 24 amostras analisadas, de cada tipo de água (sendo 12 de cada Estação de Tratamento de Água), os oocistos foram detectados em 75% das amostras de água bruta e em 12,5% das amostras de água tratada, quando estas foram concentradas por precipitação química e em 73,91% das amostras de água bruta e 33,33% das amostras das água tratada, quando as mesmas foram concentradas pela técnica da membrana filtrante. A densidade de oocistos de Cryptosporidium não apresentou correlação significativa com indicadores microbiológicos e os parâmetros físico-químicos de qualidade da água (p > 0,05). Os resultados obtidos sugeriram que o tratamento de água convencional é ineficaz para a remoção de oocistos, ressaltando a necessidade de estabelecer programas de gerenciamento em bacias hidrográficas (mananciais) que efetivamente garantam a baixa densidade de oocistos de Cryptosporidium em águas / The protozoan parasite Cryptosporidium has emerged as one of the most important contaminants of water, causing waterbone outbreaks of criptosporidiosis which have affected more than 427.000 individuals worldwide. At least 8 species of the genus Cryptosporidium has been associated with gastrointestinal disease in humans. Cryptosporidiosis can be life threatening to immunocompromised humans and can severely debilitate immunocompetent people. Cryptosporidium oocysts are environmentally robust and can survive in aquatic environments for months. Oocysts are resistant to standard chlorination disinfection used for drinking water treatment. The aims of this study were to determine the occurrence and the levels of Cryptosporidium in raw water samples and in drinking water (after flocculation, coagulation, sedimentation, filtration and disinfection) collected in two potable water treatment plants of the city of São Paulo. The relationship between parameters of water quality and the occurrence of Cryptosporidium were also analysed. Samples were collected at monthly intervals for a year. The samples were concentrated by flocculation (VESEY et alii, 1993a) and by membrane filtration (ALDOM & CHAGLA, 1994). The oocysts were identified by direct immunofluorescence assay and the presence was confirmed by phase contrast microscopy. The levels of coliforms and E.coli in the water samples were determined by the multiple tube technique using chromogenic and fluorogenic substrate (Colilert 18, Iddex). From a total of 24 samples of the each type of water investigated (12 oh the each potable water treatment plant), oocysts were detected in 75% of raw water and in 12,5% of drinking water samples, when concentrated by flocculation and in 73,91% of raw water samples and in 33,33% of drinking water samples when concentrated by membrane filtration. Cryptosporidium oocysts levels in the samples investigated was not significantly associated with microbiological indicators and water quality parameters (p > 0,05). The results suggested that the conventional treatment of water is ineffective in removing the oocysts, highlighting the necessity of establishing water catchment management programs which effectively ensure low levels of Cryptosporidium in raw water supplied to the treatment plants.

Orientador: Juliane Araújo Greinert Goulart. / Dissertação (Mestrado em Engenharia Ambiental) - Programa de Pós-Graduação em Engenharia Ambiental, Centro de Ciências Tecnológicas.

Made available in DSpace on 2016-04-28T20:16:21Z (GMT). No. of bitstreams: 1 2007- Franziska Huber.pdf: 2677706 bytes, checksum: 65e703599b63ae016e9aa85d1752e357 (MD5) Previous issue date: 2007-02-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The objectives of the present study was the genetical characterizations of Cryptosporidium spp. from different hosts, realize the sequencing an phylogenetic analysis, including the deposit in GenBank of the first Cryptosporidium sequences of animal origin, from Brazil. There were obtained fecal samples, containing Cryptosporidium oocysts from chiken, ducks, quails and Guinea pigs from a public market localized in Rio de Janeiro city, from dairy calfs maintained at a farm localized in the same city and from dogs and cats maintained at a shelter localized in the city of Nova Igua?u. For the analysis was utilized the Nested-PCR of the extracted DNA from 200μl of fecal suspension. For primary identification of Cryptosporidium species was realized RFLP with enzymes SspI and VspI. DNA samples were sequenced and phylogenetic analysis were conducted. There were diagnosed and sequenced C. baileyi infecting two ducks (DQ855339 and DQ885340) and one quail (DQ885335) and C. melagridis infecting one chicken (DQ885341). The sequences obtained form Cryptosporidium infecting Guinea pigs received accession numbers DQ885337 and DQ885338, both sequences were not identified with known Cryptosporidium species due to the great genetic distance between them and those already available at GenBank, suggesting that it may be a new genotype or species. Parasitizing cats was diagnosed C. felis (DQ885336) and in one dog C. canis (DQ885334). One sample of C. parvum of calf origin was sequenced and received accession number DQ885333. During analysis of RFLP pattern of the nested- PCR product from 18Sr DNA was stated that only C. baileyi has a characteristic digestion pattern. Other Cryptosporidium species should be digested by several other enzymes, for a accurate diagnosis. At phylogenetic analysis was found a greater genetic distance between C. felis and C. canis from Brazil when compared to the reference sequences obtained from GenBank. Based on the phylogenetic groupings, a possible new species of Cryptosporidium from Guinea Pigs calls attention for the existence of new species even in common pet animals. As is the case of the Guinea Pig. The sequences obtained in this study are the first Brazilian sequences of C. baileyi, C. meleagridis, C. felis, C. canis and C. parvum deposited in GenBank. / O presente trabalho teve por objetivo caracterizar geneticamente as esp?cies de Cryptosporidium oriundos de v?rios hospedeiros, realizar o seq?enciamento e an?lises filogen?ticas, incluindo o dep?sito das primeiras seq??ncias brasileiras de Cryptosporidium spp. de origem animal no GenBank. Foram obtidas amostras fecais contendo oocistos de Cryptosporidium de pintos, patos, codornas e porquinhos da ?ndia comercializados num mercado municipal da cidade do Rio de Janeiro, de bezerros de uma propriedade voltada ? produ??o leiteira localizada no mesmo munic?pio e de gatos e c?es de um abrigo para animais localizado no munic?pio de Nova Igua?u. Para as an?lises foi utilizado Nested-PCR do DNA extra?do a partir de 200μl de solu??o fecal. Foi realizada RFLP dos produtos obtidos no Nested-PCR, utilizando-se as enzimas SspI e VspI, para uma identifica??o preliminar das esp?cies de Cryptosporidium presentes. As amostras de DNA foram seq?enciadas e an?lises filogen?ticas foram conduzidas. Foram diagnosticados e sequenciados C. baileyi infectando dois patos (DQ855339 e DQ885340) e uma codorna (DQ885335) e C. melagridis infectando um pinto (DQ885341). As seq??ncias dos Porquinhos da ?ndia receberam os n?meros de acesso DQ885337 e DQ885338, sendo que ambas as seq??ncias n?o puderam ser identificadas como esp?cie conhecida de Cryptosporidium, devido ? grande dist?ncia gen?tica entre elas e aquelas j? depositadas no GenBank, sugerindo que se trate de um gen?ptipo ou esp?cie nova. Parasitando os gatos foi diagnosticado C. felis (DQ885336) e em um c?o C. canis (DQ885334). Uma das amostras de C. parvum de bovinos foi seq?enciada, sendo depositada no GenBank sob n?mero de acesso DQ885333. Durante as an?lises dos s?tios de corte enzim?tico dos produtos da Nested-PCR do gen 18Sr DNA, a ?nica esp?cie que realmente possue padr?o de corte caracter?stico ? C. baileyi. As demais esp?cies de Cryptosporidium deveriam ser submetidas ? a??o de outras enzimas, para um diagn?stico acurado. Nas an?lises filogen?ticas foi observada uma dist?ncia gen?tica maior entre C. felis e C. canis isolados no Brasil quando comparados ?s seq??ncias do GenBank. Com base nos dados apresentados pelo agrupamento filogen?tico, uma poss?vel nova esp?cie chama a aten??o ? presen?a de esp?cies desconhecidas de Cryptosporidium, mesmo em animais comuns de estima??o, como ? o caso do Porquinho da ?ndia. Estas s?o as primeiras seq??ncias de C. baileyi, C. meleagridis, C. felis, C. canis e C. parvum do Brasil depositadas no GenBank.

The most important priority for the management of Australian drinking water catchments is the control of pathogen loads delivered to raw water reservoirs and treatment plant intakes. A process-based mathematical model was developed to estimate pathogen catchment budgets (PCB) for Cryptosporidium, Giardia and E. coli loads generated within and exported from catchments. The model quantified key processes affecting the generation and transport of microorganisms from humans and animal excreta using land use and hydrologic data, and catchment specific information including point sources such as sewage treatment plants and on-site systems. The PCB model was applied in the Wingecarribee catchment, Sydney and used to predict and rank pathogen and indicator loads in dry weather, intermediate (<30 mm in 24 h) and large wet weather events (100mm in 24 h). Sensitivity analysis identified that pathogen excretion rates from animals and humans, and manure mobilisation rates were the most significant factors determining the output of the model. Comparison with water quality data indicated that predicted dry weather loads were generally within 1-2 log10 of the measured loads for Cryptosporidium and E. coli and within 1 log10 for Giardia. The model was subsequently used to predict and rank pathogen and indicator loads for the entire (16 000 km2) Sydney drinking water catchment.

<p>The presence of human pathogens in water indicates the sanitary risk associated with different types of water utilization. This study surveyed the sources of human pathogens in urban waters. In order to evaluate the microbiological water quality of urban water, the enumeration of various indicator bacteria (total coliform, fecal coliform, E.coli and enterococci) is usually used.</p><p>The abundance of indicator bacteria in urban water indicates the level of fecal contamination and the presence of other human pathogens such as protozoan pathogens (Giardia lamblia & Cryptosporidium parvum).</p><p>Fecal pollution of urban waters can be from human and animal origin. Point sources of fecal contamination in an urbanized area are the effluents of urban wastewater treatment plants. While non-point sources are usually originated from diffuse sources such as (runoff from roads, parking lots, pets, leaks, failing septic systems and illegal sewer connections to storm drains). urban stormwater is considered as a major carrier for delivering human pathogens from diffuse sources to receiving waters. Increases in urban stormwater volumes have resulted from increasing urbanization and growth of impervious surfaces.</p><p>In order to reduce high amounts of human pathogens in urban waters, different methods are used nowadays to develop urban wastewater treatment plants technologies and urban stormwater management practices.</p>

Cryptosporidium parvum is widely known for outbreaks within the immunocompetent population, as well its sometimes excruciating effects as an opportunistic agent in AIDS patients. Our understanding of the biology and host-parasite interactions of this parasitic protist is increasing at a rapid rate due to recent molecular and genetic advances. The topic of our research is in the area of C. parvum fatty acid metabolism, which is highly streamlined in this parasite. In addition to a type I fatty acid synthase (CpFAS1), C. parvum also possesses an enormous type I polyketide synthase (CpPKS1). Because of the size of this megasynthase, functional characterization of the complete enzyme is not possible. We have isolated and characterized the loading unit of CpPKS1 which contains an acyl-[acyl carrier protein (ACP)] ligase (AL) and an ACP. This unit is responsible for the overall substrate selection and initiation of polyketide production. Our data show that CpPKS1 prefers long-chain fatty acids with the highest specificity for arachidic acid (C20). Thus, the final polyketide product could contain as many as 34 carbons. Additionally, C. parvum possesses only a single fatty acid elongase. This family of enzymes serves a mechanism similar to FAS, and many have been found to be involved in de novo fatty acid synthesis in other organisms. After expressing this membrane protein in human cells, we have determined that it too prefers long-chain fatty acyl-CoAs which undergo only one round of elongation. This is in contrast to members of this enzyme family in other organisms that can initiate de novo synthesis from two- or four-carbon fatty acids via several rounds of elongation. Our lab has previously characterized the unique acyl-CoA binding protein (CpACBP1) from C. parvum. Molecular and biochemical data suggested that this enzyme may serve as a viable drug target. We have screened a library of known (and somewhat common) compounds against CpACBP1, and have isolated several potential compounds to be further examined for their ability to inhibit the growth of C. parvum.

A series of pilot-scale granular media filtration experiments was conducted to examine the effect of media roughness on filter performance and to evaluate the applicability of spherical, rough engineered ceramic filter media for use in granular media filters used for drinking water treatment. Filter media performance was assessed using turbidity and particle count reductions, Cryptosporidium oocyst and oocyst-sized microsphere removal, head loss and stability of operation. Experiments were designed to allow related facets of current filtration research to be examined. These included: effect of loading rate, coagulant type and dosage, and suitability of latex microspheres as surrogates for Cryptosporidium oocyst removal by granular media filtration. This study indicated that increased filter media roughness consistently improved turbidity and particle count reduction under the conditions investigated. As well, the engineered media also consistently achieved greater stability of operation during non-ideal operational periods (e.g. sudden change in filter influent turbidity).Oocyst removals were generally improved by media roughness, though this improvement was reliant on operating conditions, such as coagulant dose and type of coagulant used. The surrogate relationship between oocyst-sized latex microspheres and oocyst removal by filtration was also dependent on coagulant dose and type of coagulant. During trials with no coagulant addition, contrasts in oocyst removal were not significant, suggesting that neither surface roughness nor the size of media used were significant factors impacting oocyst removal by filtration during those periods of impaired operation. When pre-treating raw water with PACl, the engineered ceramic media achieved up to 1.25 log10 higher oocyst removals than conventional media. This improvement in oocyst removal relative to conventional media was not observed when alum was used as the primary coagulant, however. Future studies should directly compare engineered and conventional media filtration performance, using other raw water sources and different operating conditions. Biologically active filtration should also be included in future performance studies because the rough, highly porous surface of the engineered ceramic media is likely to provide excellent biofilm support.

The presence of human pathogens in water indicates the sanitary risk associated with different types of water utilization. This study surveyed the sources of human pathogens in urban waters. In order to evaluate the microbiological water quality of urban water, the enumeration of various indicator bacteria (total coliform, fecal coliform, E.coli and enterococci) is usually used. The abundance of indicator bacteria in urban water indicates the level of fecal contamination and the presence of other human pathogens such as protozoan pathogens (Giardia lamblia & Cryptosporidium parvum). Fecal pollution of urban waters can be from human and animal origin. Point sources of fecal contamination in an urbanized area are the effluents of urban wastewater treatment plants. While non-point sources are usually originated from diffuse sources such as (runoff from roads, parking lots, pets, leaks, failing septic systems and illegal sewer connections to storm drains). urban stormwater is considered as a major carrier for delivering human pathogens from diffuse sources to receiving waters. Increases in urban stormwater volumes have resulted from increasing urbanization and growth of impervious surfaces. In order to reduce high amounts of human pathogens in urban waters, different methods are used nowadays to develop urban wastewater treatment plants technologies and urban stormwater management practices.

This research aimed to address the knowledge gaps in the literature regarding the removal of waterborne pathogens (viruses and protozoa) by modified multistage slow sand filtration. In the current study, two pilot-scale multistage slow sand filtration systems were operated continuously for over two years. The pilot systems treated agricultural- and urban-impacted raw river water of variable quality with turbidity peaks over 300 NTU and seasonal cold temperatures <2°C. The first system (Pilot 1) consisted of two independent trains that included pre-ozonation, shallow-bed upflow gravel roughing filtration, and shallow-bed slow sand filtration. Pilot 1 was a pilot-scale version of an innovative, commercially available full-scale system. The second system (Pilot 2) included a full-depth upflow gravel roughing filter, a full-depth slow sand filter, and a second shallow-depth slow sand filter in series. The SSFs of both pilots were operated at high hydraulic loading rates (typically 0.4 m/h) at the upper limit of the literature recommended range (0.05 to 0.4 m/h). Both pilot systems provided excellent turbidity removal despite the high filtration rates. Effluent turbidity of all multistage SSF pilot systems were within the regulated effluent limits in Ontario for full-scale SSFs (below 1 NTU at least 95% of the time and never exceeded 3 NTU), despite raw water turbidity peaks over 100 NTU. The roughing filters contributed to approximately 60-80% of the full-train turbidity removal, compared to and 20-40% for the slow sand filters. On average, the second slow sand filter in pilot 2 provided almost no additional turbidity removal. The slow sand filter run lengths were short because of frequent high raw water turbidity, with about 50-80% of the runs in the range of 1-3 weeks. To prevent excessive SSF clogging and maintenance, filtration rates should be decreased during periods of high turbidity. Seven Cryptosporidium and Giardia challenge tests were conducted on the slow sand filters of both pilot systems at varying filtration rates (0.4 or 0.8 m/h), temperatures (2 to 25°C), and biological maturities (4 to 20 months). Removal of oocysts and cysts were good regardless of sand depth, hydraulic loading rate, and water temperature in the ranges tested. Average removals in the SSFs ranged from 2.6 to >4.4 logs for Cryptosporidium oocysts and ranged from >3.8 to >4.5 logs for Giardia cysts. This was consistent with findings in the literature, where oocyst and cyst removals of >4 logs have been reported. Cryptosporidium oocyst removals improved with increased biological maturity of the slow sand filters. At a water temperature of 2°C, average removal of oocysts and cysts were 3.9 and >4.5 logs, respectively, in a biologically mature SSF. Doubling the filtration rate from 0.4 to 0.8 m/h led to a marginal decrease in oocyst removals. Sand depths in the range tested (37-100 cm) had no major impact on oocyst and cyst removals, likely because they are removed primarily in the upper section of slow sand filter beds by straining. In general, good oocyst and cyst removals can be achieved using shallower slow sand filter bed depths and higher filtration rates than recommended in the literature. There are very few studies in the literature that quantify virus removal by slow sand filtration, especially at high filtration rates and shallow bed depths. There are no studies that report virus removal by slow sand filtration below 10°C. As such, 16 MS2 bacteriophage challenge tests were conducted at varying water temperatures (<2 to >20°C) and filtration rates (0.1 vs. 0.4 m/h) between February and June 2006 on biologically mature slow sand filters with varying bed depths (40 vs. 90 cm). Biologically mature roughing filters were also seeded with MS2. Average MS2 removals ranged from 0.2 to 2.2 logs in the SSFs and 0.1 to 0.2 logs in the RFs under all conditions tested. Virus removal by slow sand filtration was strongly dependant on hydraulic loading rate, sand depth, and water temperature. Virus removal was greater at a sand depth of 90 cm vs. 40 cm, at an HLR of 0.1 m/h vs. 0.4 m/h, and at warm (20-24°C) vs. cold (<2-10°C) water temperatures when sufficient warm water acclimation time was provided. Increased sand depth likely increased MS2 removal because of greater detention time for predation and greater contact opportunities for attachment to sand grains and biofilms. A lower HLR would also increase MS2 removal by increasing detention time, in addition to decreasing shear and promoting attachment to filter media and biofilms. Greater MS2 removal at warmer water temperatures was attributed to improved biological activity in the filters. Schmutzdecke scraping was found to have only a minor and short-term effect on MS2 removals. Virus removal can be optimized by providing deep SSF beds and operating at low filtration rates. Virus removal may be impaired in cold water, which could affect the viability of using SSF/MSF at northern climates if communities do not use disinfection or oxidation. As a stand-alone process, slow sand filtration (with or without roughing filtration) may not provide complete virus removal and should be combined with other treatment processes such as disinfection and oxidation to protect human health.