Monitoring viruses circulating in the human population and the environment is critical for protecting public and ecosystem health. The goal of this dissertation was to incorporate a viral metagenomic approach into virus surveillance efforts (both clinical and water quality control programs) to enhance traditional virus detection methods. Clinical surveillance programs are designed to identify and monitor etiological agents that cause disease. However, the ability to identify viruses may be compromised when novel or unsuspected viruses are causing infection since traditional virus detection methods target specific known pathogens. Here we describe the successful application of viral metagenomics in a clinical setting using samples from symptomatic patients collected through the Enterovirus Surveillance (EVS) program in the Netherlands (Appendix A). Despite extensive PCR-based testing, the viruses in a small percentage of these samples (n = 7) remained unidentified for more than 10 years after collection. Viral metagenomics allowed the identification of viruses in all seven samples within a week using minimal sequencing, thus rapidly filling the diagnostic gap. The unexplained samples contained BK polyomavirus, Herpes simplex virus, Newcastle disease virus and the recently discovered Saffold viruses (SAFV) which dominated the unexplained samples (n = 4). This study demonstrated that metagenomic analyses can be added as a routine tool to investigate unidentified viruses in clinical samples in a public-health setting. In addition, metagenomic data gathered for SAFV was used to complete four genotype 3 SAFV (SAFV-3) genomes through primer walking, doubling the number of SAFV-3 full genomic sequences in public databases. In addition to monitoring viruses in symptomatic patients, it is also important to monitor viruses in wastewater (raw and treated) to protect the environment from biological contamination and prevent further spread of pathogens. To gain a comprehensive understanding of viruses that endure wastewater treatment, viral metagenomics was used to survey the total DNA and RNA viral community in reclaimed water (the reusable end-product of wastewater treatment) (Appendix B). Phages (viruses that infect bacteria) dominated the DNA viral community while eukaryotic viruses similar to known plant and insect viruses dominated RNA metagenomic libraries suggesting that highly stable viruses may be disseminated through this alternative water supply. A plant virus, the Pepper mild mottle virus (PMMoV), was identified as a potential indicator of wastewater contamination based on metagenomic data and quantitative PCR assays (Appendix C). The metagenomic analysis also revealed a wealth of novel single-stranded DNA (ssDNA) viruses in reclaimed water. Further investigation of sequences with low-level similarities to known ssDNA viruses led to the completion of ten novel ssDNA genomes from reclaimed water and marine environments (Appendix D). Unique genome architectures and phylogenetic analysis suggest that these ssDNA viruses belong to new viral genera and/or families. To further explore the ecology of the novel ssDNA viruses, a strategy was developed to take metagenomic analysis to the next level by combining expression analysis and immunotechnology (Appendix E). This dissertation made a significant contribution to current microbiological data regarding wastewater by uncovering viruses that endure the wastewater treatment and identifying a new viral bioindicator.

With global freshwater supplies under pressure, viable water reuse methods are being examined to assist in improving water supplies. Municipal effluent is an ideal source for water reclamation as it is consistent in quality and quantity. The health aspects of water reuse have been identified as an issue of concern, in particular the potential presence of enteric viruses. Managed Aquifer Recharge (MAR) is a method that can aid water reclamation by recharging water such as treated effluent into a suitable aquifer. Research into the removal of pathogenic contaminants by natural processes within aquifers, namely the action of autochthonous bacteria, has led to the consideration that MAR could be used to assist in the removal of microbial pathogens. Pathogens have been demonstrated to be removed during residence in groundwater, but the presence of active autochthonous groundwater bacteria are required for significant removal rates to occur. The aim of this research was to investigate the interaction between autochthonous groundwater bacteria (AGB) and the enteroviruses Poliovirus type 1, Coxsackievirus B3 and Adenovirus B41. It was established that these viruses decrease in number in the presence of AGB but the mechanisms causing this decrease are poorly understood. Experiments were designed to examine how the individual AGB caused decay of the viruses. In this study AGB were isolated and tested for their ability in increase the decay of the viruses. It was determined that 27 % (17/63) of the isolated AGB influenced viral decay. The AGB isolates varied in their influence with only 3 out of 17 isolates being able to cause of the decay of both poliovirus and coxsackievirus. Similar variations in decay were observed for adenovirus. Decay times for all three viruses varied amongst the AGB and between the viruses. Experiments were undertaken to characterise the mechanism causing the antiviral activity of four groundwater isolates (1G, 3A, 4B and 9G) under varying conditions and treatments to give insight into the compounds or mechanisms responsible for viral decay. This would indicate whether compounds produced by the AGB responsible for viral decay were closely associated to bacterial cells (perhaps membrane bound), independent of metabolic activity, heat labile or were enzymatic in nature. The influence of enzyme inhibiters and heat treatment indicated that viral degradation is caused by compounds that are enzymatic in nature. As viral numbers were monitored by nucleic acid copy numbers rather than via infectivity assays, the viral protein coats must be the first step in degradation followed by the removal of the viral nucleic acid. This two step process would require both protease and nuclease enzymes to result in loss of viral numbers as measured by RT-PCR/PCR. Further characterisation and identification of these four bacterial isolates was also carried out. Three out of the four isolates were sequenced and analysed using partial 16S rRNA gene sequences to determine their phylogenetic relationships compared to related organisms. Isolate 3A was placed in the order Burkholderiales. Isolate 4B was placed in the family Xanthomonadaceae. Isolate 9G was placed in the family Rhizobiaceae. Isolate 1G was only partially sequenced and preliminary identification placed it in the phylum Bacteriodetes. Understanding of the processes carried out by AGB within an aquifer during MAR using reclaimed waters will aid in increasing the viability of this water reuse process. If important natural processes could be utilised to remediate any potential pathogens, the health concerns with reclaimed waters could be addressed and solved simply through prescribed retention times within the aquifer. Key species of AGB may even be utilised as markers to assess the suitability of an aquifer for MAR.

A diarréia em bezerros é comprovadamente uma das principais causas de perdas econômicas em rebanhos de corte, sendo os prejuízos diretamente derivados da redução do ganho de peso e custos com tratamento. Considerando-se que, para o delineamento de medidas profiláticas adequadas ao controle de uma doença é necessário o conhecimento de suas causas, o presente trabalho teve por objetivo estudar de modo longitudinal a freqüência de ocorrência de coronavírus bovino (BCoV), rotavírus do grupo A (RV-A), protozoários, helmintos e enterobactérias em uma propriedade com casos esporádicos e em outra com casos recorrentes de diarréia em bezerros. Investigou-se, ainda, a presença de genes codificadores de para as toxinas shiga-like 1 e 2 , para as adesinas K99 e F41 e para a intimina (eae) nas E. coli isoladas. Para a detecção de BCoV, utilizou-se uma nested-RT-PCR direcionada ao gene codificador da RNA polimerase RNA dependente, e para a detecção de RV-A utilizou-se ELISA direto duplo sanduíche. O isolamento e caracterização bioquímica das colônias de bactérias foram conduzidos de acordo com técnicas de bacteriologia clássica. A caracterização genotípica das colônias de E. coli isoladas foi realizada através de uma Multiplex-PCR, utilizando primers para os genes codificadores dos seguintes fatores de virulência: Stx1, Stx2, Sta, K99, F41 e eae. A detecção de parasitas foi realizada por centrífugo sedimentação em água-eter e centrífugo-flutuação em solução supersaturada de sacarose, considerando-se como resultado final do exame coproparasitológico todos os parasitas encontrados em ambas as técnicas em sua maior contagem. A freqüência de ocorrência de coronavírus bovino e rotavírus do grupo A foi baixa, restrita a animais com até quatro meses de idade, enquanto que protozoários, helmintos e enterobactérias apresentaram tendência crescente em freqüência relacionada à progressão da idade dos animais. Foram identificados os patotipos STEC, AEEC e um padrão atípico com os genes de K99 e eae. Além disso, encontrou-se associação entre baixo padrão sanitário e maior freqüência de ocorrência de Eimeria spp, Strongyloidea, Strongyloides spp, Trichuris spp e Escherichia coli portadora de fatores de virulência Stx1, Stx2, K99 e eae. O presente trabalho colabora para a caracterização da microbiota entérica de bezerros, mas mantém abertas as questões relativas às diferenças específicas entre as biotas entéricas de bezerros sadios e com diarréia, enfatizando a importância de se realizarem mais estudos que investiguem simultaneamente diversos enteropatógenos potenciais nestes animais / Diarrhea in calves is proved to be one of the main causes of economic loss in beef herds, which is direct originated by reduction of weight gain and increment of treatment costs. It is known that the determination of appropriate preventive measures to control a disease depends on the knowledge of its causes. In this manner, the present work aimed to study longitudinally the frequency of occurrence of bovine coronavirus (BCoV), group A rotavirus (RV-A), protozoa, helminths and enterobacteria in a beef farm presenting sporadic cases and other with recurrent cases of diarrhea. Moreover, the presence of genes that express the following virulence factors were investigated in the isolated E. coli: shiga-like toxins 1 and 2, adhesins K99 and F41 and intimin (eae). A nested-RT-PCR targeted to the RNA-dependent RNA polimerase gene was conducted to detect BCoV, and a double-sandwich direct ELISA was used to detect RV-A. Classical bacteriology techniques were used to isolate and indentify bacterial colonies. A Multiplex-PCR, with primers designed to previously described E. coli genes, was used to genotype these colonies. Centrifuge sedimentation in water-ether and centrifuge flotation in sucrose supersaturated solution were used to detect the presence of parasites. The final result was achieved by the highest count of all the parasites found. The observed frequency of occurrence of both BCoV and RV-A was low and restricted to animals up to 4 months of age whereas protozoa, helminths and enterobacteria showed an increasing tendency on its frequency related to progression of animals aging. STEC, AEEC and an atypical pathotype of E. coli, with K99 and eae genes were found. Moreover, an association between low sanitary pattern and a higher frequency of Eimeria spp, Strongyloidea, Strongyloides spp, Trichuris spp and Escherichia coli bearing Stx1, Stx2, K99 and eae genes was observed. The present study contributes to the characterization of calf enteric microbiota. However, the points related to the specific differences between healthy and diarrheic calves enteric microbiota remain unclear, emphasizing the importance of future studies to investigate different potential enteropathogens in beef calves simultaneously

Viruses are common causes of both endemic and epidemic gastroenteritis, infecting millions of people per year, with norovirus, rotavirus and adenovirus-F as the main causative agents, and sapovirus and astrovirus as contributing viruses. These viruses are highly infectious and most severe in the very young, old, or individuals who are immunocompromised. The viral infection usually causes self-limited gastroenteritis, although chronic infection has been observed in highly immunocompromised patients. African and South-East Asian regions are disproportionally affected by diarrhoeal disease. These regions (especially South Africa) are also more severely affected by human immunodeficiency virus (HIV) infections. It has been suggested that immunocompromised individuals may form part of a reservoir for novel virus variants and recombinants. It should be taken into account that not every person is equally susceptible to infection after pathogen exposure and that not all infected persons develop clinical symptoms (Ramani and Giri, 2019). One host genetic factor that can influence susceptibility to enteric infection is the expression of histo-blood group antigens (HBGAs). Histo-blood group antigens are a major group of complex carbohydrates and are determinants of both human and animal ABO blood groups and the Lewis blood group systems, which are distributed in abundance on the mucosal epithelia of the gastrointestinal tract. Histo-blood group antigens have been proven to influence susceptibility to rotavirus and norovirus infections. Saliva, blood and stool specimens (n=205) have previously been collected from children (≤ 5 years of age) hospitalised with gastroenteritis at Kalafong Provincial Tertiary Hospital from June 2016 to December 2017. Follow up stool specimens were then collected six weeks after enrolment when possible. A descriptive questionnaire was completed by each child’s guardian, giving information on age, residential area, HIV status etc. of the participating child. The stool specimens were screened for six gastroenteritis causing viruses (norovirus GI and –GII, rotavirus, sapovirus, astrovirus and adenovirus) by multiplex PCR. Forty-seven percent (96/205) of specimens tested positive for at least one gastroenteritis causing virus. Rotavirus predominated (46/205), followed by norovirus (32/205), adenovirus (15/205), sapovirus (9/205) and astrovirus (3/205). A total of 27/32 norovirus (GI.3, GII.2, GII.3, GII.4, GII.7, GII.12 and GII.21), 44/46 rotavirus (G1P[8], G2P[4], G2P[6], G3P[4], G3P[8], G8P[4], G8P[6], G9P[6] and G9P[8]) and 8/9 sapovirus (GI.1, GI.2, GII.1, GII.4 and GII.8) strains have been genotyped, of which norovirus GII.4 and rotavirus G3P[4] predominated. A total of 46/205 children submitted a follow up stool specimen to be tested. Of the 46 children, 9 tested positive for norovirus infection with initial stool specimen testing. Follow up screening resulted in 13/46 (28%) specimens testing positive for either norovirus GI or GII, with all patients presenting as asymptomatic. After genotyping it was observed that only one of the follow up specimens were identical to the original sequence genotyped, indicating prolonged shedding. FUT2 genotyping of 205/205 children showed a 71%:29% ratio between secretors and non-secretors. Eighty percent (77/96) of the virus-infected children were secretors whereas only 20% (19/96) were non-secretors. Rotavirus (p<0.01) and norovirus GII.4 (p<0.05) specifically were found to be more prevalent in secretors. In this study, no statistical significance was observed in terms of severity of and susceptibility to gastroenteritis viruses between HIV-infected, HIV-exposed uninfected or HIV-uninfected individuals. Histo-blood group phenotyping has resulted in various combinations, with Le(b) being the most prevalent antigen found. Next generation sequencing was unsuccessful. In future, fresh specimens should be considered for testing, with more funding and time for optimisation of this process and to give adequate results. In summary, gastroenteritis is still a leading cause of childhood morbidity and mortality, with all advancements in understanding the disease helping to decrease the impact of it. This study again reinforced the importance of these viruses, as they are circulating in such high abundance. It also reinforced the concept that susceptibility to noro- and rotavirus infection is affected by the secretor status of a person. This could in future help with better understanding the viral infection mechanisms and in turn help with vaccine development and treatment / Dissertation (MSc (Medical Virology))--University of Pretoria, 2020. / Reese Mushrooms / Discovery grant / PRF / Medical Virology / Msc / Unrestricted

Les virus entérotropes sont des virus ubiquitaires infectant une large catégorie de vertébrés dont l’homme et les primates non-humains (PNHs). Ils se transmettent principalement par voie féco-orale directe ou indirecte à la suite de laquelle ils atteignent les entérocytes et s’y multiplient. Bien que parfois asymptomatiques, les infections causées par les virus entérotropes peuvent se manifester par des gastroentérites très fréquentes chez les enfants de moins de 5 ans. Ces mêmes virus peuvent être responsables de pathologies sévères telles que les maladies respiratoires, encéphalitiques, cardiaques, neurologiques. À partir des années 1950, de nombreux virus entérotropes ont été isolés de tissus de PNHs couramment utilisés en cultures cellulaires et en recherche biomédicale. Dès lors, de nombreuses études ont été conduites sur la caractérisation des virus entérotropes principalement chez les PNHs captifs ou en contact avec l’homme. En milieu naturel, en dehors des entérovirus et des adénovirus, leur circulation, leur épidémiologie et leur diversité restent encore peu connues. L’objectif de cette thèse est donc de rechercher et caractériser les virus entérotropes chez les PNHs d’Afrique Centrale. Ainsi à partir de 600 échantillons de fèces de PNHs collectés dans des forêts et réserves naturelles au Gabon, nous avons pu mettre en évidence la circulation de différentes espèces d’entérovirus (EVs) chez les mandrills et les chimpanzés. Cette caractérisation a également permis de mettre en évidence des EVs proches d’EVs infectant l’homme ainsi que deux nouveaux sérotypes chez un chimpanzé et chez un mandrill. Nous avons également mis en évidence un astrovirus (AstV) totalement divergent d’AstVs référencés chez un gorille. En dehors de leur circulation en milieu naturel, les virus entérotropes sont également présents chez les PNHs en contact fréquents avec l’homme. De ce fait à partir d’échantillons fécaux d’un groupe de 12 chimpanzés du Sanctuaire de Tchimpounga, nous avons caractérisé l’EV-C99 responsable de cas de paralysie chez l’homme et probablement responsable de celle observée chez un chimpanzé. De plus, deux sapovirus (SaVs) très proches d’un SaV identifié chez l’homme ont également été caractérisés. L’Afrique Centrale est donc caractérisée par une diversité de virus entérotropes qui circulent chez les PNHs. L’identification chez les PNHs de virus entérotropes proches en milieu naturel de ceux infectant l’homme soulève l’existence d’une probabilité de transmission inter-espèce entre les PNHs et l’homme dont le sens reste encore à déterminer. Par contre chez les PNHs du sanctuaire, la susceptibilité à ces virus humains peut être responsable de pathologies graves comme la paralysie observée chez les chimpanzés. / The enteric viruses are ubiquitous virus infecting a broad range of vertebrates, including humans and non-human primates (NHPs). They are spread by direct or indirect fecal-oral route following which they reach the enterocytes and multiply. Even though infections caused by these viruses are asymptomatic, enteric viruses could be responsible for frequent gastroenteritis in children under 5 years of age. These viruses may be responsible for severe pathologies such as respiratory, encephalitic, cardiac and neurological diseases. In the 1950s, many viruses have been isolated from NHPs species commonly used in cell culture and biomedical research. Since, many studies have been conducted to characterize, then enteric viruses have been mainly identified in captive NHPs or those living in close contact with humans. Little is known concerning the circulation, epidemiology and diversity of enteric viruses in the wild, except for enteroviruses and adenoviruses. The objective of this thesis is to investigate and characterize the enteric virus in NHPs of Central Africa. Thus from 600 samples of feces of NHPs collected in natural forests and reserves in Gabon, we highlighted the circulation of different species enteroviruses (EVs) in mandrills and chimps. We also identified EVs close to those infecting humans as well as two new serotypes in a chimpanzee and in a mandrill. We have highlighted an astrovirus (AstV) completely divergent from those referenced in a gorilla. Apart from their outstanding natural environment, enteric viruses are also present in NHPs in frequent contact with humans. Therefore fecal samples from a group of 12 chimpanzees from the Tchimpounga Sanctuary, we characterized the EV-C99 responsible for cases of paralysis in humans and probably responsible for that observed in a chimpanzee. In addition, two sapovirus (SaVs) very close to a SaV identified in humans have also been characterized. Central Africa is therefore characterized by a diversity of enteric virus circulating in NHPs. The identification in the wild of enteric virus in NHPs close to those infecting humans raises probability of cross-species transmissions between NHPs and humans whose sense remains to be determined. However in NHPs in the sanctuary, susceptibility to these human viruses can be responsible for severe diseases such as paralysis observed in chimpanzee.

M. Tech (Department of Biotechnology, Faculty of Applied and Computer Sciences) Vaal University of Technology. / The presence of enteric viruses in drinking water poses a health risk to consumers. It is therefore very important for drinking water suppliers to provide water that is pathogen free and fit for human consumption. This can be achieved by an effective water treatment system that ensures the safety of water from the treatment plant until the water reaches the consumer. This study assessed the ability of a conventional water treatment system to remove viruses. The system consisted of three unit processes, namely, clarification, sand filtration and disinfection. These processes were simulated on a bench-scale to determine the effectiveness of each one at removing viruses. Clarification was conducted using a Phipps and Bird jar testing system and three different chemical treatments: (i) Polyelectrolyte (SUDFLOC 3835), (ii) a combination of lime and activated silica and (iii) a combination of lime, activated silica and ferric chloride. Sand filtration was simulated using a Phipps and Bird column filtration system. Disinfection was conducted using free chlorine. The findings from this study showed that the removal or inactivation of viruses increased with an increase in the concentration of chemicals added. For clarification, the combination of lime, activated silica and ferric chloride was the most effective treatment for the removal or inactivation of viruses. Sand filtration was found to be ineffective for the removal of viruses. Disinfection was shown to be the most effective process for the removal or inactivation of viruses. While clarification, sand filtration and disinfection did not remove or inactivate viruses equally, the entire treatment chain is still essential. This is because even if a barrier does not directly remove viruses it ensures that subsequent processes can function effectively. Overall the treatment processes should not be considered as discrete barriers but rather an integrated system that must function throughout to avoid a risk to customers.

O objetivo deste estudo foi avaliar a qualidade virológica da água de reúso produzida em uma das estações de tratamento de esgoto da cidade de São Paulo. Para tanto, foram coletadas concomitantemente 177 amostras de esgoto tratado (100L) e bruto (15L) e os vírus concentrados utilizando método Viradel-ultracentrifugação. Em seguida as amostras foram tratadas com Vertrel XF e os ácidos nucléicos extraídos para a detecção de adenovírus (HAdV), rotavírus (RV-A), norovírus e vírus da hepatite A (VHA). A detecção por PCR e/ou RT-PCR evidenciou RV-A (G1-G5), VHA e HAdV incluindo os da espécie F tanto no esgoto bruto quanto no tratado, no entanto norovírus não foram detectados em ambos os efluentes. A infectividade de RV-A e HAdV foi avaliada por cultivo celular e os rotavírus RV-A foram também quantificados por reação de imunoperoxidase direta. PCR em tempo real foi padronizada para quantificação de vírus não cultiváveis ou de difícil cultivo como os VHA. Com base nos resultados obtidos foi verificada a ocorrência e a distribuição anual de cada vírus nas águas de reúso. / The aim of the study was to evaluate the virological quality from one Sewage Treatment Plant in the state of São Paulo. From January/2005 to November/2006, 177 samples (15L) of raw sewage were collected at the entrance and another 177 (100L) at the end of treatment, twice a week. Viruses were concentrated by filtration through positively charged microporous filters, followed by ultracentrifugation. Virus concentrates were treated by using Vertrel-XF and the viral genomes extracted for detection of adenovirus (HAdV), rotavirus (RV-A), norovirus and hepatitis A virus (HAV). PCR and RT-PCR revealed RV-A (G1-G5), HAV and HAdV, including the enteric ones (species F) in sewage and treated wastewater samples. Norovirus was not detected in any samples. The infectivity of HAdV and RV-A was assayed by inoculating onto suitable cell line. Immunoperoxidase assay was used to calculate the rotavirus FFU/L in the samples. Real time-PCR was standardized for enumeration of non-cultivable virus. The occurrence and annual distribution of each virus in reuse water were analyzed.

Introdução - A prática de reúso pela aplicação de esgotos tratados na agricultura atualmente é considerada parte integrante dos recursos hídricos por muitas nações. No entanto, oferece riscos à saúde da população pela presença de bactérias, cistos de protozoários, ovos de helmintos e vírus, capazes de sobreviver por longos períodos em ambientes desfavoráveis e apresentarem um curto tempo entre a infecção e o desenvolvimento da doença. Objetivo - Estabelecer medidas preventivas para o lançamento de efluentes nos ecossistemas aquáticos, controle sanitário na redução de patógenos transmitidos por águas contaminadas e a disposição de efluentes tratados em solos tropicais como ferramentas de auxílio do manejo integrado dos recursos hídricos em bacias hidrográficas, por meio do uso de metodologia de gerenciamento de risco com aplicação do Sistema APPCC, em um conjunto de tecnologias na produção de efluente tratado de esgoto de origem doméstica para avaliar o potencial de emprego em solos agrícolas. Métodos - A pesquisa foi desenvolvida na estação de tratamento de esgoto e no campo experimental para pesquisas de utilização de efluente na agricultura no município de Lins. Os indicadores biológicos estudados e os pontos de amostragem foram determinados através da utilização da metodologia de controle de qualidade Análise de Perigos e Pontos Críticos de Controle (APPCC). Foram coletadas amostras do solo irrigado e dos poços de monitoramento do lençol freático. As amostras foram submetidas a análises físico-químicas, microbiológicas e parasitológicas. Aos resultados obtidos foi aplicada a Avaliação Quantitativa do Risco Microbiológico, para a determinação do risco anual de infecção. Resultados - O efluente final apresentou concentrações médias de 8,13x105 NMP/100 mL de coliformes totais e 4,69x105 NMP/100 mL de Escherichia coli, não sendo observados ovos de helmintos. O solo apresentou maior concentração de coliformes totais e E. coli na camada superficial 1,01x106 e 8,70x103, diminuindo ao longo da profundidade. Os ovos de helmintos foram encontrados entre a superfície e os 15 cm de profundidade em concentrações entre 0,07 e 0,87 ovos/g de peso seco. Foi constatada a presença de adenovírus em concentração de 3,56x105 cópias genômicas/g entre os 5 e 10 cm de profundidade em 2015. Nos poços de monitoramento foram constatadas concentrações de coliformes totais variando entre < 1,00 e 1,01x103 NMP/100 mL e E. coli entre <1,00 e 1,00x100 NMP/100 mL. Não foi observada a presença de vírus entéricos. O risco anual de infecção estimado para E. coli no efluente final foi 1,02x10-2 pppa, no solo variou entre 2,68x10-3 e 7,59x10-3 pppa e nos poços de monitoramento variou entre 1,42x10-8e 4,38x10-9 pppa. Para os ovos de helmintos observados no efluente do tratamento primário o risco anual de infecção calculado foi 4,04x10-2 pppa e no solo variou entre 9,57x10-1 e 9,76x10-1 pppa. Conclusões No período da pesquisa, as amostras dos sistemas de tratamento de esgoto e de irrigação demonstraram atender as expectativas de remoção de patógenos condizentes aos seus parâmetros de projeto, havendo restrições para seu lançamento em corpos hídricos e aplicação na agricultura. O solo mostrou capacidade de retenção dos organismos patogênicos nos primeiros 60 cm de profundidade, reduzindo as suas concentrações a níveis inferiores a 1,00 NMP/100 mL nos aquíferos. / Introduction - Currently, the practice of reuse through the application of treated sewage in agriculture considers an integral part of water resources for many nations. However, threatens the health of the population by the presence of bacteria, protozoan cysts, helminth eggs and viruses able to survive for long periods in harsh environments and present a short time between infection and development of disease. Objective - Establish preventive measures for the release of effluents on aquatic ecosystems, sanitary control in the reduction of pathogens transmitted by contaminated water and the disposal of treated effluent in tropical soils as aid tools in integrated management of water resources in river basins. For this purpose it was employed as risk management methodology the Hazard Analysis and Critical Control Points system (HACCP) in a set of technologies in the treated effluent production of domestic sewage to assess the employment potential in agricultural soils. Methods - The study was conducted in the sewage treatment plant and in an adjoining experimental field situated at Lins city. The biological indicators and the sampling points were determined by using the quality control methodology Hazard Analysis and Critical Control Points (HACCP). Samples of irrigated soil and groundwater monitoring wells were collected. The samples were subjected to analysis, physical-chemical, microbiological and parasitological. The results obtained was applied to Quantitative Microbial Risk Assessment (QRMA), for determining the annual risk of infection. Results - The final effluent presented average concentrations of 8.13x105 MPN / 100 mL of total coliforms and 4.69x105 MPN / 100 mL of Escherichia coli, not being observed helminth eggs. The soil showed the highest concentration of total coliforms and E. coli in the surface layer and 1.01x106 8.70x103, decreasing along the depth. Eggs from helminths were found between the surface and 15 cm depth in concentrations from 0.07 to 0.87 eggs / g dry weight. It was found the presence of adenovirus in concentration 3.56x105 genomic copies / g between 5 and 10 cm depth in 2015. In monitoring wells total coliform concentrations were found ranging from <1.00 and 1.01x103 NMP / 100 mL and E. coli between <1.00 and 1.00x100 MPN / 100 mL. It was not observed the presence of enteric viruses. The estimated annual risk of infection to E. coli in the final effluent was 1.02x10-2 pppy, soil ranged from 2.68x10-3 and 7.59x10-3 pppy and monitoring wells ranged from 1.42x10-8 e 4.38x10-9 pppy. For helminth eggs observed in the effluent from the primary treatment, the annual risk of infection was calculated 4.04x10-2 pppy and soil ranged from 9.57x10-1 and 9.76x10-1 pppy. Conclusions - During the survey period, samples of sewage and irrigation treatment systems showed meet the expectation removal of pathogens consistent to its design parameters, with restrictions on its release in water and applied in agriculture bodies. The soil of pathogenic organisms showed retention capacity in the first 60 cm depth by reducing its concentration to levels below 1.00 MPN / 100 mL in aquifers.

Made available in DSpace on 2016-12-23T14:04:29Z (GMT). No. of bitstreams: 1 Sara Angelino Martins.pdf: 1671026 bytes, checksum: 54e5627bbd7b0f309897c96d6bc54a06 (MD5) Previous issue date: 2013-06-10 / A região da Ilha das Caieiras, na cidade de Vitória (ES) situa-se dentro de uma região estuarina na Baía de Vitória e encontra-se próxima a uma extensa área de manguezal. A população residente mantém relação de grande dependência com os recursos naturais oferecidos (ostras, mariscos) que são utilizados como fonte de subsistência e lazer. O manguezal da Ilha das Caieiras recebe esgoto in natura proveniente das áreas adjacentes, contaminando a água e os mariscos ali existentes. Além disso, os microrganismos que são lançados juntamente com o esgoto no ambiente costeiro podem ser acumulados no sedimento. O presente trabalho teve por objetivo pesquisar vírus entéricos e bactérias indicadoras de poluição fecal no sedimento e na água em três pontos do estuário (P1, P2 e P3) na área de manguezal, ao longo de 15 meses de monitoramento. Foram avaliadas um total de 45 amostras para presença de coliformes termotolerantes (CT), enterococos e vírus entéricos (adenovírus HAdV; rotavírus RVA; e norovírus NoV GII), utilizando-se a Nested-PCR. Também foram avaliados parâmetros físico-químicos da água próxima ao ponto de coleta do sedimento. As análises microbiológicas do sedimento apresentaram respectivamente as seguintes médias geométricas para enterococos: 1,5x103, 1,5x103 e 1,1x103 UFC/100g, coliformes termotolerantes: 4,9x102, 2,6x102, 5,1x102. Nas amostras de água os resultados encontrados para enterococos foram: 7,6x101, 5,0x101 e 1,1x102 e coliformes termotolerantes 2,2x102, 1,3x102, 3,0x102. As análises de vírus entéricos nas amostras de água apresentaram frequências de 20% para RV, 13% para AdV e 8,8% para NoV GII e nas amostras de sedimento somente NoV GII(4,5%) foi encontrado. A presença viral e bacteriológica nas amostras indica que esta região encontra-se sob impacto antropogênico, e que o sedimento proveniente do manguezal da Ilha das Caieiras pode atuar como reservatório de substâncias poluentes que ao serem liberadas para a coluna d agua podem causar doenças gastrointestinais às pessoas que utilizam essa área como fonte de subsistência e lazer / The region of the Ilha das Caieiras, in Vitória (ES) is located within an estuarine region in Vitoria Bay and is close to an extensive mangrove area. The resident population ratio remains highly dependent relationship on natural resources offered (oysters, clams) therefore used as a source of subsistence and recreation. The mangrove of Ilha das Caieiras receives sewage from adjacent areas, contaminating water and shellfish therein. In addition, microorganisms that are cast together with the sewage in the coastal environment can be accumulated in the sediment. This objective of this work was to investigate enteric viruses and bacterial indicators of fecal pollution in the sediment and water at three points of this estuary (P1, P2 and P3) mangrove area over 15 months of monitoring. We evaluated a total of 45 samples for the presence of thermotolerant coliform (TC), enterococci and enteric viruses (adenovirus - HAdV; rotavirus - RVA, and norovirus - NoV GII, using a nested PCR. We also evaluated the physico-chemical parameters of the water collection point next to the sediment. Microbiological analyzes of the sediment were respectively geometric mean for enterococci: 1.5 x103, 1.5 x103 and 1.1 x103 UFC/100g, thermotolerantes coliforms: 4.9 x103, 2.6 x102 and 5.1 x102. In water samples the results for enterococci were: 7.6 x101, 5.0 x101 and 1.1 x102 and themortolerants coliforms: 2.2 x102, 1.3 x102, and 3.0 x102. Analyses of enteric viruses in water samples showed frequencies of 20% for RV, 13% for AdV and 8.8% for NoV GII and sediment samples only NoV GII (4.5%) was found. The presence of the viral and bacteriological samples indicates that this region is under anthropogenic impact, and that the sediment from the mangrove of Ilha das Caieiras can act as a reservoir of pollutants that are released to the water column can cause gastrointestinal diseases the people who use this area as a source of livelihood and recreation

Introdução - A prática de reúso pela aplicação de esgotos tratados na agricultura atualmente é considerada parte integrante dos recursos hídricos por muitas nações. No entanto, oferece riscos à saúde da população pela presença de bactérias, cistos de protozoários, ovos de helmintos e vírus, capazes de sobreviver por longos períodos em ambientes desfavoráveis e apresentarem um curto tempo entre a infecção e o desenvolvimento da doença. Objetivo - Estabelecer medidas preventivas para o lançamento de efluentes nos ecossistemas aquáticos, controle sanitário na redução de patógenos transmitidos por águas contaminadas e a disposição de efluentes tratados em solos tropicais como ferramentas de auxílio do manejo integrado dos recursos hídricos em bacias hidrográficas, por meio do uso de metodologia de gerenciamento de risco com aplicação do Sistema APPCC, em um conjunto de tecnologias na produção de efluente tratado de esgoto de origem doméstica para avaliar o potencial de emprego em solos agrícolas. Métodos - A pesquisa foi desenvolvida na estação de tratamento de esgoto e no campo experimental para pesquisas de utilização de efluente na agricultura no município de Lins. Os indicadores biológicos estudados e os pontos de amostragem foram determinados através da utilização da metodologia de controle de qualidade Análise de Perigos e Pontos Críticos de Controle (APPCC). Foram coletadas amostras do solo irrigado e dos poços de monitoramento do lençol freático. As amostras foram submetidas a análises físico-químicas, microbiológicas e parasitológicas. Aos resultados obtidos foi aplicada a Avaliação Quantitativa do Risco Microbiológico, para a determinação do risco anual de infecção. Resultados - O efluente final apresentou concentrações médias de 8,13x105 NMP/100 mL de coliformes totais e 4,69x105 NMP/100 mL de Escherichia coli, não sendo observados ovos de helmintos. O solo apresentou maior concentração de coliformes totais e E. coli na camada superficial 1,01x106 e 8,70x103, diminuindo ao longo da profundidade. Os ovos de helmintos foram encontrados entre a superfície e os 15 cm de profundidade em concentrações entre 0,07 e 0,87 ovos/g de peso seco. Foi constatada a presença de adenovírus em concentração de 3,56x105 cópias genômicas/g entre os 5 e 10 cm de profundidade em 2015. Nos poços de monitoramento foram constatadas concentrações de coliformes totais variando entre < 1,00 e 1,01x103 NMP/100 mL e E. coli entre <1,00 e 1,00x100 NMP/100 mL. Não foi observada a presença de vírus entéricos. O risco anual de infecção estimado para E. coli no efluente final foi 1,02x10-2 pppa, no solo variou entre 2,68x10-3 e 7,59x10-3 pppa e nos poços de monitoramento variou entre 1,42x10-8e 4,38x10-9 pppa. Para os ovos de helmintos observados no efluente do tratamento primário o risco anual de infecção calculado foi 4,04x10-2 pppa e no solo variou entre 9,57x10-1 e 9,76x10-1 pppa. Conclusões No período da pesquisa, as amostras dos sistemas de tratamento de esgoto e de irrigação demonstraram atender as expectativas de remoção de patógenos condizentes aos seus parâmetros de projeto, havendo restrições para seu lançamento em corpos hídricos e aplicação na agricultura. O solo mostrou capacidade de retenção dos organismos patogênicos nos primeiros 60 cm de profundidade, reduzindo as suas concentrações a níveis inferiores a 1,00 NMP/100 mL nos aquíferos. / Introduction - Currently, the practice of reuse through the application of treated sewage in agriculture considers an integral part of water resources for many nations. However, threatens the health of the population by the presence of bacteria, protozoan cysts, helminth eggs and viruses able to survive for long periods in harsh environments and present a short time between infection and development of disease. Objective - Establish preventive measures for the release of effluents on aquatic ecosystems, sanitary control in the reduction of pathogens transmitted by contaminated water and the disposal of treated effluent in tropical soils as aid tools in integrated management of water resources in river basins. For this purpose it was employed as risk management methodology the Hazard Analysis and Critical Control Points system (HACCP) in a set of technologies in the treated effluent production of domestic sewage to assess the employment potential in agricultural soils. Methods - The study was conducted in the sewage treatment plant and in an adjoining experimental field situated at Lins city. The biological indicators and the sampling points were determined by using the quality control methodology Hazard Analysis and Critical Control Points (HACCP). Samples of irrigated soil and groundwater monitoring wells were collected. The samples were subjected to analysis, physical-chemical, microbiological and parasitological. The results obtained was applied to Quantitative Microbial Risk Assessment (QRMA), for determining the annual risk of infection. Results - The final effluent presented average concentrations of 8.13x105 MPN / 100 mL of total coliforms and 4.69x105 MPN / 100 mL of Escherichia coli, not being observed helminth eggs. The soil showed the highest concentration of total coliforms and E. coli in the surface layer and 1.01x106 8.70x103, decreasing along the depth. Eggs from helminths were found between the surface and 15 cm depth in concentrations from 0.07 to 0.87 eggs / g dry weight. It was found the presence of adenovirus in concentration 3.56x105 genomic copies / g between 5 and 10 cm depth in 2015. In monitoring wells total coliform concentrations were found ranging from <1.00 and 1.01x103 NMP / 100 mL and E. coli between <1.00 and 1.00x100 MPN / 100 mL. It was not observed the presence of enteric viruses. The estimated annual risk of infection to E. coli in the final effluent was 1.02x10-2 pppy, soil ranged from 2.68x10-3 and 7.59x10-3 pppy and monitoring wells ranged from 1.42x10-8 e 4.38x10-9 pppy. For helminth eggs observed in the effluent from the primary treatment, the annual risk of infection was calculated 4.04x10-2 pppy and soil ranged from 9.57x10-1 and 9.76x10-1 pppy. Conclusions - During the survey period, samples of sewage and irrigation treatment systems showed meet the expectation removal of pathogens consistent to its design parameters, with restrictions on its release in water and applied in agriculture bodies. The soil of pathogenic organisms showed retention capacity in the first 60 cm depth by reducing its concentration to levels below 1.00 MPN / 100 mL in aquifers.