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ABSTRACT: The kinetics of ozone inactivation of infectious prion protein (PrP(Sc), scrapie 263K) was investigated in ozone-demand free phosphate buffered saline (PBS). Diluted infectious brain homogenates (IBH) (0.01%) were exposed to pre-determined ozone dose (10.8 ± 2.0 mg/L) at three pHs (pH 4.4, 6.0 and 8.0) and two temperatures (4(o)C and 20°C). The inactivation of PrP(Sc) was quantified by determining the in vitro destruction of PrP(Sc) templating properties using the protein misfolding cyclic amplification (PMCA) assay and bioassay, which were shown to correlate well. The inactivation kinetics were characterized by both Chick-Watson (CW) and efficiency factor Hom (EFH) models. It was found that the EFH model fit the experimental data more appropriately. The efficacy of ozone inactivation of PrP(Sc) was both pH and temperature dependent. Based on the EFH model, CT values were determined for 2-log(10), 3-log(10), and 4-log(10) inactivation at the conditions they were achieved. Our results indicated that ozone is effective on prion inactivation in ozone-demand free water and may be applied for the inactivation of infectious prion in prion contaminated water and wastewater.
Applied and environmental microbiology 02/2013; · 3.69 Impact Factor
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ABSTRACT: Macrophages are immune cells that play a pivotal role in the detection and elimination of pathogenic
microorganisms. Macrophages possess a variety of surface receptors devoted to the recognition of
non-self by discriminating between host and pathogen-derived structures. Recognition of foreign
microorganisms by the macrophage ultimately results in phagocytosis and eventual destruction of
microorganisms by lysosomal enzymes, toxic reactive oxygen and nitrogen intermediates, and/or
nutrient deprivational mechanisms. This review focuses on the cellular processes involved in
macrophage-mediated host defense; from the initial host-pathogen interactions that mediate
recognition, to the mechanisms employed by macrophages to destroy and eliminate the pathogen.
02/2013; , ISBN: 978-57808-327-5
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Romain Marti,
Victor P J Gannon,
Cassandra Jokinen,
Martin Lanthier,
David R Lapen, Norman F Neumann,
Norma J Ruecker,
Andrew Scott,
Graham Wilkes,
Yun Zhang,
Edward Topp
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ABSTRACT: Over a seven-year period (2004-2010) 1095 water samples were obtained from the South Nation River basin at multiple watershed monitoring sites (Ontario, Canada). Real-time PCR using Bacteroidales specific markers was used to identify the origin (human (10% prevalence), ruminant (22%), pig (∼2%), Canada goose (4%) and muskrat (7%)) of fecal pollution. In parallel, the distribution of fecal indicator bacteria and waterborne pathogens (Cryptosporidium oocysts, Giardia cysts, Escherichia coli O157:H7, Salmonella enterica and Campylobacter spp.) was evaluated. Associations between the detection of specific Bacteroidales markers and the presence of fecal indicator bacteria, pathogens, and distinct land use or environmental variables were evaluated. Linear correlations between Bacteroidales markers and fecal indicator bacteria were weak. However, mean marker densities, and the presence and absence of markers could be discriminated on the basis of threshold fecal indicator densities. The ruminant-specific Bacteroidales marker was the most frequently detected marker in water, consistent with the large number of dairy farms in the study area. Detection of the human or the ruminant markers were associated with a slightly higher risk of detecting S. enterica. Detection of the muskrat marker was related to more frequent Campylobacter spp. detections. Important positive associations between markers and pathogens were found among: i) total Bacteroidales and Cryptosporidium and Giardia, ii) ruminant marker and S. enterica, and iii) muskrat and Campylobacter spp.
Water Research 02/2013; · 4.86 Impact Factor
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ABSTRACT: Nearly 690 raw surface water samples were collected during a 6 year period from multiple watersheds in the South Nation River basin, Ontario Canada. Cryptosporidium oocysts in water samples were enumerated, sequenced and genotyped by detailed phylogenetic analysis. The resulting species and genotypes were assigned to broad known host and human infection risk classes. Wildlife/unknown, livestock, avian, and human host classes occurred in 21, 13, 3, and <1% of sampled surface water, respectively. Cryptosporidium andersoni was the most commonly detected livestock species, while Muskrat I and II genotypes were the most dominant wildlife genotypes. The presence of Giardia spp., Salmonella spp., Campylobacter spp., and E. coli O157:H7 was evaluated in all water samples. The greatest (significant) odds of Giardia spp., Campylobacter spp. and Salmonella spp. in water was associated, respectively, with the presence of livestock (odds ratio (OR) =3.1), avian (OR=4.3), and livestock (OR=9.3) host classes. Classification and regression tree analyses (CART) were used to group generalized host and human infection risk classes on the basis of a broad range of environmental and land use variables, while tracking co-occurrence of zoonotic pathogens in these groupings. The occurrence of livestock associated Cryptosporidium was most strongly related to agricultural water pollution in the fall (conditions also associated with elevated odds of other zoonotic pathogens occurring in water), whereas wildlife/unknown sources of Cryptosporidium were geospatially associated with smaller watercourses where urban/rural development was relatively lower. Conditions that support wildlife may not necessarily increase overall human infection risks associated with Cryptosporidium, since most Cryptosporidium classed as wildlife in this study (e.g., Muskrat I and II genotype) do not pose significant infection risks to humans. Consequently, from a human health perspective, land use practices in agricultural watersheds that create opportunities for wildlife to flourish, should not be refuted solely on their potential to increase relative proportions of wildlife fecal contamination in surface water. The present study suggests that mitigating livestock fecal pollution in surface water in this region would likely reduce human infection risks associated with Cryptosporidium and other zoonotic pathogens.
Applied and environmental microbiology 11/2012; · 3.69 Impact Factor
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ABSTRACT: Environmental concentrations of Cryptosporidium require molecular assays with ultra-sensitive detection limits and which provide critical information on genetic diversity within the genus, a feat particularly challenging from a diagnostics point of view. In this study, the performance of repetitive nested PCR-RFLP and limiting template dilution repetitive nested PCR-RFLP were assessed for their ability to detect Cryptosporidium and resolve mixtures of species and genotypes on microscope slides prepared by USEPA Method 1623 from raw water samples. Seventy percent of water samples positive for Cryptosporidium oocysts by immunofluorescent microscopy tested positive by molecular assays and resulted in species/genotype identification. Multiple species/genotypes were detected in 41% of the samples, including 30 samples from which 3 species/genotypes were detected and 11 samples where 4 species/genotypes were detected. In all, 29 species or genotypes were detected which were represented by the 102 different sequences identified. Of these, 64 were considered novel as no matches were available in GenBank. These results support the use of repetitive and limiting template approaches for the detection and resolution of Cryptosporidium from the environment as well as further supporting the use of DNA sequencing as the most appropriate tool for identifying Cryptosporidium species and genotypes from water.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 10/2012; · 3.22 Impact Factor
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ABSTRACT: The high sequence diversity and heterogeneity observed within species or genotypes of Cryptosporidium requires phylogenetic approaches for the identification of novel sequences obtained from the environment. A long-term study on Cryptosporidium in the agriculturally-intensive South Nation River watershed in Ontario, Canada was undertaken, in which 60 sequence types were detected. Of these sequence types 33 were considered novel with no identical matches in GenBank. Detailed phylogenetic analysis identified that most sequences belonged to 17 previously described species: Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium ubiquitum, Cryptosporidium meleagridis, muskrat I, muskrat II, deer mouse II, fox, vole, skunk, shrew, W12, W18, W19 and W25 genotypes. In addition, two new genotypes were identified, W27 and W28. C. andersoni and the muskrat II genotype were most frequently detected in the water samples. Species associated with livestock made up 39% of the total molecular detections, while wildlife associated species and genotypes accounted for 55% of the Cryptosporidium identified. The human pathogenic species C. hominis and C. parvum had an overall prevalence of 1.6% in the environment, indicating a small risk to humans from the Cryptosporidium present in the watershed. Phylogenetic analysis and knowledge of host-parasite relationships are fundamental in using Cryptosporidium as a source-tracking or human health risk assessment tool.
Water Research 07/2012; 46(16):5135-50. · 4.86 Impact Factor
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ABSTRACT: Misfolded prions (PrP(Sc)) are well known for their resistance to conventional decontamination processes. The potential risk of contamination of the water environment, as a result of disposal of specified risk materials (SRM), has raised public concerns. Ozone is commonly utilized in the water industry for inactivation of microbial contaminants and was tested in this study for its ability to inactivate prions (263K hamster scrapie = PrP(Sc)). Treatment variables included initial ozone dose (7.6 to 25.7 mg/liter), contact time (5 s and 5 min), temperature (4°C and 20°C), and pH (pH 4.4, 6.0, and 8.0). Exposure of dilute suspensions of the infected 263K hamster brain homogenates (IBH) (0.01%) to ozone resulted in the in vitro destruction of the templating properties of PrP(Sc), as measured by the protein misfolding cyclic amplification (PMCA) assay. The highest levels of prion inactivation (≥4 log(10)) were observed with ozone doses of 13.0 mg/liter, at pH 4.4 and 20°C, resulting in a CT (the product of residual ozone concentration and contact time) value as low as 0.59 mg · liter(-1) min. A comparison of ozone CT requirements among various pathogens suggests that prions are more susceptible to ozone degradation than some model bacteria and protozoa and that ozone treatment may be an effective solution for inactivating prions in water and wastewater.
Applied and environmental microbiology 12/2011; 78(3):613-20. · 3.69 Impact Factor
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ABSTRACT: Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.
Applied and environmental microbiology 06/2011; 77(12):3998-4007. · 3.69 Impact Factor
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ABSTRACT: In developed countries, clean and safe drinking water is taken for granted, and water treatment processes used to mass-produce drinking water have been hailed as one of the top five engineering achievements of the 20th century. However, in the last decade, several waterborne disease outbreaks have made us painfully aware of the personal, economical, societal, and public health costs associated with the impact of waterborne disease. There is evidence to suggest that the prevalence of waterborne disease may be dramatically underestimated in developed countries and that routine endemic exposure to waterborne pathogens may occur more frequently than originally perceived. A variety of demographical, societal, environmental, and physiological emergence factors likely play critical roles in enhancing the frequency of transmission of pathogens to hosts. This review focuses on the scope and impact of waterborne disease in developed countries and identifies issues surrounding the potential for rapid global emergence or re-emergence of waterborne disease. We examine relevant literature on Cryptosporidium parvum and Escherichia coli O157 as model organisms of study that support these concepts. Key words: drinking water, treatment, public health, Cryptosporidium, Escherichia coli, emergence, pathogens, transmission, waterborne disease.
Journal of Environmental Engineering and Science. 02/2011; 4(3):155-171.
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ABSTRACT: Temporal evolution of microbiological, physical, and chemical quality of stormwater runoff from a stormwater drain in an urban residential area in Calgary, Canada, was investigated from May to September, 2006 and 2007. Investigating event mean concentrations and their correlations with rainfall characteristics revealed that intensive rainfall events produced highly polluted stormwater runoff when pollutant source limitation did not occur. Inconsistent event-based correlations between total suspended solids (TSS) concentrations and water quality parameters were observed. During storms, the loading of TSS exhibited a flow-dependent nature, whereas microorganism discharge appeared to be governed by a flow-independent mechanism. No strong first-flush effect was observed in either TSS or microorganisms, on average. No correlations of first-flush loads of TSS with rainfall characteristics were identified. Moderate negative correlations between first-flush loads of microorganisms and rainfall depth and intensity indicated that first flush of microorganisms tended to occur in small storms.
Water Environment Research 12/2010; 82(12):2333-45. · 0.88 Impact Factor
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ABSTRACT: This research investigates the potential impacts of climate change on stormwater quantity and quality generated by urban residential areas on an event basis in the rainy season. An urban residential stormwater drainage area in southeast Calgary, Alberta, Canada is the focus of future climate projections from general circulation models (GCMs). A regression-based statistical downscaling tool was employed to conduct spatial downscaling of daily precipitation and daily mean temperature using projection outputs from the coupled GCM. Projected changes in precipitation and temperature were applied to current climate scenarios to generate future climate scenarios. Artificial neural networks (ANNs) developed for modelling stormwater runoff quantity and quality used projected climate scenarios as network inputs. The hydrological response to climate change was investigated through stormwater runoff volume and peak flow, while the water quality responses were investigated through the event mean value (EMV) of five parameters: turbidity, conductivity, water temperature, dissolved oxygen (DO) and pH. First flush (FF) effects were also noted. Under future climate scenarios, the EMVs of turbidity increased in all storms except for three events of short duration. The EMVs of conductivity were found to decline in small and frequent storms (return period < 5 years); but conductivity EMVs were observed to increase in intensive events (return period ≥ 5 years). In general, an increasing EMV was observed for water temperature, whereas a decreasing trend was found for DO EMV. No clear trend was found in the EMV of pH. In addition, projected future climate scenarios do not produce a stronger FF effect on dissolved solids and suspended solids compared to that produced by the current climate scenario. Copyright © 2010 John Wiley & Sons, Ltd.
Hydrological Processes 11/2010; 25(8):1298 - 1312. · 2.49 Impact Factor
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ABSTRACT: Recent molecular evidence suggests that different species and/or genotypes of Cryptosporidium display strong host specificity, altering our perceptions regarding the zoonotic potential of this parasite. Molecular forensic profiling of the small-subunit rRNA gene from oocysts enumerated on microscope slides by U.S. Environmental Protection Agency method 1623 was used to identify the range and prevalence of Cryptosporidium species and genotypes in the South Nation watershed in Ontario, Canada. Fourteen sites within the watershed were monitored weekly for 10 weeks to assess the occurrence, molecular composition, and host sources of Cryptosporidium parasites impacting water within the region. Cryptosporidium andersoni, Cryptosporidium muskrat genotype II, Cryptosporidium cervine genotype, C. baileyi, C. parvum, Cryptosporidium muskrat genotype I, the Cryptosporidium fox genotype, genotype W1, and genotype W12 were detected in the watershed. The molecular composition of the Cryptosporidium parasites, supported by general land use analysis, indicated that mature cattle were likely the main source of contamination of the watershed. Deer, muskrats, voles, birds, and other wildlife species, in addition to sewage (human or agricultural) may also potentially impact water quality within the study area. Source water protection studies that use land use analysis with molecular genotyping of Cryptosporidium parasites may provide a more robust source-tracking tool to characterize fecal impacts in a watershed. Moreover, the information is vital for assessing environmental and human health risks posed by water contaminated with zoonotic and/or anthroponotic forms of Cryptosporidium.
Applied and Environmental Microbiology 07/2007; 73(12):3945-57. · 3.83 Impact Factor
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ABSTRACT: The emerging concept of host specificity of Cryptosporidium spp. was exploited to characterize sources of fecal contamination in a watershed. A method of molecular forensic profiling of Cryptosporidium oocysts on microscope slides prepared from raw water samples processed by U.S. Environmental Protection Agency Method 1623 was developed. The method was based on a repetitive nested PCR-restriction fragment length polymorphism-DNA sequencing approach that permitted the resolution of multiple species/genotypes of Cryptosporidium in a single water sample.
Applied and Environmental Microbiology 01/2006; 71(12):8991-4. · 3.83 Impact Factor
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ABSTRACT: Cryptosporidium is a faecally transmitted parasite and an aetiologic agent of human enteric disease worldwide. Contaminated food, water, and direct contact with infected animals have long been considered risk factors for parasitic infection. The applications of molecular diagnostic tools in clinical and environmental research settings have inspired a new understanding of Cryptosporidium taxonomy and disease transmission. Once thought to be a non-specific parasite with a broad host range, cryptosporidia are now considered to be relatively host specific, bringing into question the true zoonotic potential of this parasite. Contaminated water is considered a major risk factor contributing to outbreaks of human cryptosporidiosis. In light of Cryptosporidium host-specificity and the expanding complexity associated with the molecular epidemiology of this parasite, the ability to differentiate the various species and genotypes of Cryptosporidium from environmental water samples becomes imperative. These emerging concepts need to be integrated into the risk assessment strategies used to associate the public health impact of water supplies contaminated with Cryptosporidium spp.
Reviews in Medical Microbiology 12/2005; 17(1):1-9. · 0.37 Impact Factor
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ABSTRACT: Macrophages are immune cells that play a pivotal role in the detection and elimination of pathogenic microorganisms. Macrophages possess a variety of surface receptors devoted to the recognition of non-self by discriminating between host and pathogen-derived structures. Recognition of foreign microorganisms by the macrophage ultimately results in phagocytosis and the eventual destruction of microorganisms by lysosomal enzymes, toxic reactive oxygen and nitrogen intermediates, and/or nutrient deprivational mechanisms. However, protozoan parasites such as Toxoplasma gondii, Trypanosoma cruzi, and Leishmania spp., parasitize macrophages, utilizing them as a host cell for their growth, replication, and/or maintenance of their life cycles. The protozoan parasites of the genus Leishmania are unique in that their intracellular replication in the host is predominantly restricted to a single cell type, the macrophage. This review focuses on the cellular processes involved in macrophage-mediated host defense against protozoan parasites, from the initial host-parasite interactions that mediate recognition to the mechanisms employed by macrophages to destroy and eliminate the pathogen. As an example model system of experimental study, we describe in more more detail the cellular interactions between macrophages and the obligate intracellular parasite of mammalian macrophages, Leishmania spp.
Critical Reviews in Microbiology 02/2002; 28(3):187-248. · 6.27 Impact Factor
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OZONE SCIENCE AND ENGINEERING. 01/2001; 23(1):1-13.
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ABSTRACT: In vitro excystation is often used as a measure of viability of encysted protozoan parasites. Parasites that do not excyst in vitro are assumed to be non-viable and non-infectious, whereas those that do excyst are assumed viable. To test the validity of these assumptions, Cryptosporidium parvum oocysts were excysted in vitro using two different excystation protocols, and the non-excysted intact oocysts were isolated using flow cytometry. Non-excysted sorted oocysts readily infected neonatal CD-1 mice. Increasing the duration of the excystation assays from 1 h to 3 h resulted in a higher percent of excysted oocysts, but the remaining non-excysted parasites were still capable of infecting neonatal CD-1 mice. Our results suggest that in vitro excystation is not an accurate measure of the viability or infectious potential of C. parvum oocysts.
FEMS Microbiology Letters 01/2000; 183(2):331 - 336. · 2.04 Impact Factor
