Enteric pathogens have a substantial impact on human health as they can cause outbreaks and severe disease outcomes. These pathogens employ many virulence strategies to evade host defenses and cause disease. While some virulence strategies have been carefully studied, other mechanisms remain largely uncharacterized. In addition, there are a number of putative virulence factors that have yet to be phenotypically or biochemically characterized. In order to facilitate the development of novel and effective treatment strategies for enteric pathogens, an understanding of how these putative virulence-associated proteins contribute to pathogenesis is required. In this work, the characterizations of two proteins implicated in the processes of motility and type III secretion are presented. The Escherichia coli O157:H7 protein Z0021 is found in an O-island unique to the most virulent serotypes of Shiga-toxin producing E. coli. Z0021 was found to encode for a repressor of motility that exerted its regulatory effect prior to the activation of class II promoters in the flagellar cascade. This work provided the first identification and characterization of a fimbrial operon-encoded motility repressor in E. coli O157:H7. The second protein, SsaN, from Salmonella enterica is a putative type III secretion system ATPase. This work examined the role of SsaN in virulence and effector secretion, and moreover provided insight into the mechanism by which SsaN binds to a chaperone to facilitate effector secretion. Together, these findings contribute to the understanding of the virulence strategies of two enteric pathogens that have had, and continue to have, significant impacts on human health worldwide. / Thesis / Doctor of Philosophy (PhD)

During the harvesting process, meat products can become contaminated with enteric pathogens, such as Escherichia coli O157:H7 and Salmonella Typhimurium. Surrogates for these pathogens would be beneficial for validating carcass decontamination treatments. Surrogate organisms are organisms that behave similarly to specific pathogens but are non-pathogenic and can be used to determine efficacy of decontamination regimes for pathogens. The surrogates proposed are non-pathogenic, ampicillin-resistant E. coli biotype I strains that were previously isolated from beef cattle hides. Each E. coli strain was transformed to express a fluorescent protein (red: EcRFP; green: EcGFP; yellow: EcYFP) that is detectable under an ultraviolet light source. Surface areas on hot boned beef carcasses (clod, brisket, outside round) were inoculated with a fecal slurry containing EcRFP, EcGFP, EcYFP and rifampicin-resistant E. coli O157:H7 and S. Typhimurium. Surface regions were then treated in a model spray cabinet using an initial water wash (28ºC) followed by treatments using 2% L-lactic acid (55ºC), hot water (95ºC at source) or a combination of the two. Treatments were compared for their effectiveness at reducing populations of inoculated (4.7 to 6.7 log CFU/cm2) E. coli, S. Typhimurium, EcRFP, EcGFP and EcYFP. Log reductions for inoculated organisms were calculated individually and then total and average surrogate cocktail values were calculated. All decontamination treatments reduced the inoculated numbers of pathogens and surrogates to near or below the detection limit of 0.5 log CFU/cm2. The combined treatment resulted in the greatest log reductions. The three individual surrogate organisms varied in log reductions according to the different decontamination treatments applied; however, log reductions for the total surrogate cocktail did not differ significantly from that of E. coli O157:H7. With the exception of EcYFP, the individual surrogates and average surrogate cocktail were significantly more resistant to microbial interventions including lactic acid than S. Typhimurium. Because abattoirs utilize different carcass decontamination treatments, it is difficult for one single fluorescent protein-producing isolate to accurately represent the behavior of E. coli O157:H7 or S. Typhimurium. Instead, surrogates should be used as a total cocktail to accurately represent the effectiveness of different treatments for reduction of enteric pathogens.

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A cross-sectional study investigated the shedding of zoonotic organisms (Campylobacter, Giardia, and Salmonella) and antimicrobial resistant generic E. coli in dogs that visited dog parks in southern Ontario. Logistic regression models were constructed to identify risk factors. Factors for the shedding of Campylobacter spp. included consumption of a commercial dry diet, exposure to compost, and age. Factors for the shedding of C. upsaliensis included outdoor water access and age. A risk factor for ampicillin resistance was attending a dog day care. For resistance to at least one antimicrobial, factors included attending a dog day care, breed size, consumption of a commercial dry diet and consumption of a homemade cooked diet. For multiclass resistance, exposure to compost, breed size, and consumption of a commercial dry diet were identified. Park was not significant in any model. Dogs that visit dog parks shed organisms that may pose a human health risk. / Canadian Institutes of Health Research (CIHR) Institute of Population and Public Health/ Public Health Agency of Canada Applied Public Health Research Chair awarded to J. M. Sargeant; Public Health Agency of Canada; Ontario Veterinary College Pet Trust Fund; and a grant to D. L. Pearl from the Canada Foundation for Innovation and the Ontario Research Fund.

No description available.

Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2017-08-30T13:20:01Z No. of bitstreams: 2 Deisi_Tapparo2017.pdf: 1334800 bytes, checksum: e1f7fae75650df8c87e6f134d2dad8cf (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-30T13:20:01Z (GMT). No. of bitstreams: 2 Deisi_Tapparo2017.pdf: 1334800 bytes, checksum: e1f7fae75650df8c87e6f134d2dad8cf (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The swine breeding stands out on worldwide due to its constant growth. However, environmental problems have increased as the activity gains recognition, consequently, planning and management of waste produced have been required in the systems. Animal carcasses disposal inside or outside the animal rearing farms is under concern and object of discussion about good practices to do it considering biosecurity aspects. One alternative is the swine manure and swine carcass co-digestion in order to improve biogas production. At the same time, it is known that animal carcass has some pathogenic microorganisms of zoonotic importance. In this context, this research aimed at studying swine carcass co-digestion and swine manure as well as its implications on biogas yield and digestate sanitation. This study was carried out under mesophilic temperature (37 ºC) and triplicate tests. Biogas volume was measured using eudiometer tubes, according to VDI 4630. The trials used the carcass sample digestion in a separate way and loading rates of carcass/manure were 3, 7.5 and 15kg.m-3manure, which represented 1, 2.5 and 5 times of mortality/manure production rates on typical swine farms (Mortality rate of 7% .year-1 for matrices). The inactivation trials were carried out in separated. Then it was evaluated the inactivation of the following microorganisms models (E. coli, Salmonella enterica serovar Senftenberg, PCV2, and bacteriophage MS2 and PhiX-174). Four inactivation strategies were carried out at two temperatures (24 ºC and 37 ºC) and two swine carcass/swine manure ratios were also studied (3kgcarcass.m-3manure and 15 kgcarcass.m-3manure). The swine carcass presented biochemical methane potential (BMP) of 1076 ± 48 LNbiogas.kgSVadic-1, and its co-digestion with swine manure increases biogas production potential when compared to manure monodigestion. The model microorganisms such as E. coli, S. Senftenberg and PCV2 (37°C) were completely inactivated until 30 days, while PCV2 (24°C), MS2 and PhiX-174 were more resistant. The temperature of co-digestion process influences the time required for microorganisms’ inactivation. Inactivation results suggest that, during co-digestion at 37°C, there is a greater pathogen reduction when compared to the same process at 24° C. It is recommended to use a pre-treatment process to biodigestor without heating system and under mesophilic temperature to ensure biosafety. / A suinocultura se destaca no cenário mundial devido ao seu constante crescimento. Todavia, os problemas ambientais aumentam à medida que a atividade ganha reconhecimento, por isso, são necessários planejamento e gestão dos resíduos produzidos nos sistemas. Um dos desafios da atividade é a destinação de animais mortos, logo, são necessárias alternativas que aliem proteção do meio ambiente e saúde pública bem como opções de baixo custo. Uma das alternativas é realizar a codigestão de carcaça suína com dejeto suíno para melhorar a produção de biogás. Ao mesmo tempo, é de conhecimento que a carcaça animal possui micro-organismos patogênicos de importância zoonótica. Nesse contexto, o objetivo desta pesquisa foi estudar a codigestão de dejeto suíno e carcaça suína, suas implicações sobre a produção de biogás e sanitização do digestato. Os estudos de digestão foram conduzidos em batelada, sob condições mesofílicas (37ºC) e em triplicata. Foram utilizados tubos eudiômetros para mensurar o biogás produzido, conforme norma VDI 4630. Os experimentos envolveram a digestão de amostras de carcaça em separado e das relações de mistura entre carcaça e dejeto nas seguintes proporções: 3; 7,5 e 15 kgcarcaça.m-3dejeto, as quais representaram 1; 2,5 e 5 vezes a mortalidade encontrada em granjas comerciais (mortalidade de 7%.ano-1 para matrizes). Os experimentos de inativação de patógenos foram conduzidos em separado. Avaliou-se a inativação de micro-organismos modelos (E. coli, Salmonella entérica serovar Senftenberg, PCV2, MS2 e PhiX-174). Quatro estratégias de inativação foram estudadas: duas temperaturas (24ºC e 37ºC) e duas relações de carcaça suína/dejeto suíno (3kg.m-3 e 15kg.m-3). Os resultados mostraram que a carcaça suína tem um potencial de 1076 ± 48 LNbiogás.kgSVadic-1, e a sua codigestão com dejeto suíno aumenta o potencial de produção de biogás comparando com a monodigestão do dejeto. Os micro-organismos modelo E. coli, S. Senftenberg e PCV2 (37 ºC) foram totalmente inativados em 30 dias, enquanto o PCV2 (24 ºC), MS2 e PhiX -174 foram mais resistentes. A temperatura do processo de codigestão influencia no tempo necessário para a inativação dos micro-organismos. Os resultados de inativação sugerem que, durante a codigestão a 37 ºC, ocorre maior redução de patógenos quando comparada ao mesmo processo a 24 ºC. Recomenda-se a utilização de processo de pré-tratamento para biodigestores sem sistema de aquecimento e em temperaturas mesofílicas, a fim de garantir a biossegurança.

<p>Greywater is domestic household wastewater without inputfrom the toilet, i.e. wastewater from sinks, the shower,washing machine and dishwasher in a home. Source separation ofgreywater can be a strategy to enhance recirculation of plantnutrients and/or improve water use. The risk for transmissionof disease when reusing greywater is largely dependent on thecross-contamination by faeces. High levels of faecalindicators, mainly thermotolerant coliform bacteria, have beenreported in greywater, indicating substantial faecal pollution.However, growth of indicator bacteria within the system leadsto an overestimation of thefaecal input and thus the hygienerisk. The faecal input of the greywater in Vibyåsen,Sollentuna, North of Stockholm, was estimated to be 0.04 ±0.02 g faeces person-1 day-1 from the quantification of thefaecal sterol coprostanol, compared to 65 g, 5.2 g and 0.22 gp-1 d-1 using E. coli, enterococci and cholesterolrespectively.</p><p>Prevalence of pathogens in the population and the faecalload based on coprostanol concentrations were used to form thebasis of a screening-level quantitative microbial riskassessment (QMRA) that was undertaken for rotavirus, Salmonellatyphimurium, Campylobacter jejuni, Giardia intestinalis andCryptosporidium parvum, looking at the treatment required to bebelow an acceptable level of risk (10-3) for reuse or dischargeof the greywater. The different exposure scenarios simulated–groundwater recharge, direct contact, irrigation andrecreational water–showed that a reduction of 0.7–3.7 log was needed for rotavirus, with the measured level offaecal load in Vibyåsen. The other pathogen of concern wasCampylobacter, where a 2.2 log reduction was needed forgroundwater recharge. The infectious dose of Salmonella is highand the excretion numbers of Giardia cysts and Cryptosporidiumoocysts low, resulting in no treatment requirements for theseorganisms under these circumstances. Pathogen input fromcontaminated food via the kitchen sink had a minor effect onthe microbiological quality of the greywater. Studies on virusoccurrence in greywater as well as validation of the faecalload of greywater at another site would give valuable input forfuture QMRAs.</p><p>Greywater treatment efficiency studies, especially on virusremoval, are scarce and more investigations are warranted.Active sludge may not be a suitable technique for greywater dueto the low carbon content in this flow. Chemical precipitationhas the advantage of removing phosphorus as well as virusesefficiently and it is suggested as one possible method fortreating greywater. Otherwise the most common practice forgreywater treatment in Sweden is soil infiltration. However, itis suggested that the recommendations for wastewaterinfiltration also be observed for greywater, despite the lowfaecal load, due to the simulated results on virus reductionneeded.</p><p><b>Key words:</b>greywater, greywater reuse, greywatertreatment, microbial risk assessment, groundwater recharge,irrigation, recreational water, faecal contamination, indicatorbacteria, index organisms, faecal sterols, bacteriophages,enteric pathogens, rotavirus, Salmonella, Campylobacter,Giardia, Cryptosporidium, Legionella</p>

Human excreta contain plant nutrients and have the potentialto be used as a fertiliser in agriculture. Urine contributesthe major proportion of the nutrients (N, P and K) in domesticwastewater whereas faeces contribute a smaller amount andinvolves greater health risks if reused due to the possiblepresence of enteric pathogens. Human urine does not generallycontain pathogens that can be transmitted through theenvironment. Source-separation of urine and faeces is possible by usingurine-separating (or urine-diverting) toilets, available assimple dry toilets or porcelain flush toilets with dividedbowls. The risk for transmission of disease when handling andreusing the urine is largely dependent on thecross-contamination by faeces. In this research, the presenceof human faeces in urine samples was successfully determined byanalysing for faecal sterols. Cross-contamination was evidentin 22% of the samples from urine collection tanks, and in thesequantified to an average (± SD) of 9.1 ± 5.6 mgfaeces per litre urine. Testing for indicator bacteria wasshown to be an unsuitable method for determining faecalcontamination in human urine sinceE. colihad a rapid inactivation in the urine and faecalstreptococci were found to grow within the system. The fate of any enteric pathogens present in urine iscrucial for the risk for transmission of infectious diseases.Gram-negative bacteria (e.g.SalmonellaandE. coli) were rapidly inactivated (time for 90%reduction, T90&lt;5 days) in source-separated urine at itsnatural pH-value of 9. Gram-positive faecal streptococci weremore persistent with a T90of approximately 30 days. Clostridia sporenumbers were not reduced at all during 80 days. Similarly,rhesusrotavirus andSalmonella typhimuriumphage 28B were not inactivated inurine at low temperature (5°C), whereas at 20°C theirT90-values were 35 and 71 days, respectively.Cryptosporidiumoocysts were less persistent with a T90of 29 days at 4°C. Factors that affect thepersistence of microorganisms in source-separated human urineinclude temperature, pH, dilution and presence of ammonia. By using Quantitative Microbial Risk Assessment (QMRA), therisks for bacterial and protozoan infections related tohandling and reuse of urine were calculated to be&lt;10-3for all exposure routes independent of the urinestorage time and temperature evaluated. The risk for viralinfection was higher, calculated at 0.56 for accidentalingestion of 1 ml of unstored urine. If the urine was stored at20°C for 6 months the risk for viral infection was reducedto 5.4 × 10-4. By following recommendations for storage and reuse, whichare dependent on the type of crop to be fertilised, it ispossible to significantly decrease the risk for infections. Sofar, the level of risk that is acceptable is unknown. Theacceptable risk will be one of the main factors determining thefuture utilisation of source-separated human urine inagriculture. <b>Keywords:</b>urine-separation, urine, wastewater systems,wastewater reuse, recycling, enteric pathogens, faecal sterols,indicator bacteria, hygiene risks, microbial persistence,microbial risk assessment, QMRA, fertiliser, crop.

<p>Human excreta contain plant nutrients and have the potentialto be used as a fertiliser in agriculture. Urine contributesthe major proportion of the nutrients (N, P and K) in domesticwastewater whereas faeces contribute a smaller amount andinvolves greater health risks if reused due to the possiblepresence of enteric pathogens. Human urine does not generallycontain pathogens that can be transmitted through theenvironment.</p><p>Source-separation of urine and faeces is possible by usingurine-separating (or urine-diverting) toilets, available assimple dry toilets or porcelain flush toilets with dividedbowls. The risk for transmission of disease when handling andreusing the urine is largely dependent on thecross-contamination by faeces. In this research, the presenceof human faeces in urine samples was successfully determined byanalysing for faecal sterols. Cross-contamination was evidentin 22% of the samples from urine collection tanks, and in thesequantified to an average (± SD) of 9.1 ± 5.6 mgfaeces per litre urine. Testing for indicator bacteria wasshown to be an unsuitable method for determining faecalcontamination in human urine since<i>E. coli</i>had a rapid inactivation in the urine and faecalstreptococci were found to grow within the system.</p><p>The fate of any enteric pathogens present in urine iscrucial for the risk for transmission of infectious diseases.Gram-negative bacteria (e.g.<i>Salmonella</i>and<i>E. coli</i>) were rapidly inactivated (time for 90%reduction, T₉₀<5 days) in source-separated urine at itsnatural pH-value of 9. Gram-positive faecal streptococci weremore persistent with a T₉₀of approximately 30 days. Clostridia sporenumbers were not reduced at all during 80 days. Similarly,<i>rhesus</i>rotavirus and<i>Salmonella typhimurium</i>phage 28B were not inactivated inurine at low temperature (5°C), whereas at 20°C theirT₉₀-values were 35 and 71 days, respectively.<i>Cryptosporidium</i>oocysts were less persistent with a T₉₀of 29 days at 4°C. Factors that affect thepersistence of microorganisms in source-separated human urineinclude temperature, pH, dilution and presence of ammonia.</p><p>By using Quantitative Microbial Risk Assessment (QMRA), therisks for bacterial and protozoan infections related tohandling and reuse of urine were calculated to be<10^-3for all exposure routes independent of the urinestorage time and temperature evaluated. The risk for viralinfection was higher, calculated at 0.56 for accidentalingestion of 1 ml of unstored urine. If the urine was stored at20°C for 6 months the risk for viral infection was reducedto 5.4 × 10^-4.</p><p>By following recommendations for storage and reuse, whichare dependent on the type of crop to be fertilised, it ispossible to significantly decrease the risk for infections. Sofar, the level of risk that is acceptable is unknown. Theacceptable risk will be one of the main factors determining thefuture utilisation of source-separated human urine inagriculture.</p><p><b>Keywords:</b>urine-separation, urine, wastewater systems,wastewater reuse, recycling, enteric pathogens, faecal sterols,indicator bacteria, hygiene risks, microbial persistence,microbial risk assessment, QMRA, fertiliser, crop.</p>