Distributions of fecal markers in wastewater from different climatic zones for human fecal pollution tracking in Australian surface waters

Recreational and potable water supplies polluted with human wastewater can pose a direct health risk to humans. Therefore, sensitive detection of human fecal pollution in environmental waters is very important to water quality authorities around the globe. Microbial source tracking (MST) utilizes human fecal markers (HFMs) to detect human wastewater pollution in environmental waters. The concentrations of these markers in raw wastewater are considered important because it is likely that a marker whose concentration is high in wastewater will be more frequently detected in polluted waters. In this study, quantitative PCR (qPCR) assays were used to determine the concentrations of fecal indicator bacteria (FIB) Escherichia coli and Enterococcus spp., HFMs Bacteroides HF183, human adenoviruses (HAdVs), and polyomaviruses (HPyVs) in raw municipal wastewater influent from various climatic zones in Australia. E. coli mean concentrations in pooled human wastewater data sets (from various climatic zones) were the highest (3.2 × 10 gene copies per ml), followed by those of HF183 (8.0 × 10 gene copies per ml) and Enterococcus spp. (3.6 × 10 gene copies per ml). HAdV and HPyV concentrations were 2 to 3 orders of magnitude lower than those of FIB and HF183. Strong positive and negative correlations were observed between the FIB and HFM concentrations within and across wastewater treatment plants (WWTPs). To identify the most sensitive marker of human fecal pollution, environmental water samples were seeded with raw human wastewater. The results from the seeding experiments indicated that Bacteroides HF183 was more sensitive for detecting human fecal pollution than HAdVs and HPyVs. Since the HF183 marker can occasionally be present in nontarget animal fecal samples, it is recommended that HF183 along with a viral marker (HAdVs or HPyVs) be used for tracking human fecal pollution in Australian environmental waters

Comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters

Pathogenic human viruses cause over half of gastroenteritis cases associated with recreational water use worldwide. They are relatively difficult to concentrate from environmental waters due to typically low concentrations and their small size. Although rapid enumeration of viruses by quantitative PCR (qPCR) has the potential to greatly improve water quality analysis and risk assessment, the upstream steps of capturing and recovering viruses from environmental water sources along with removing PCR inhibitors from extracted nucleic acids remain formidable barriers to routine use. Here, we compared the efficiency of virus recovery for three rapid methods of concentrating two microbial source tracking (MST) viral markers human adenoviruses (HAdVs) and polyomaviruses (HPyVs) from one liter tap water and river water samples on HA membranes (90 mm in diameter). Samples were spiked with raw sewage, and viral adsorption to membranes was promoted by acidification (method A) or addition of MgCl2 (methods B and C). Viral nucleic acid was extracted directly from membranes (method A), or viruses were eluted with NaOH and concentrated by centrifugal ultrafiltration (methods B and C). No inhibition of qPCR was observed for samples processed by method A, but inhibition occurred in river samples processed by B and C. Recovery efficiencies of HAdVs and HPyVs were ∼10-fold greater for method A (31 to 78%) than for methods B and C (2.4 to 12%). Further analysis of membranes from method B revealed that the majority of viruses were not eluted from the membrane, resulting in poor recovery. The modification of the originally published method A to include a larger diameter membrane and a nucleic acid extraction kit that could accommodate the membrane resulted in a rapid virus concentration method with good recovery and lack of inhibitory compounds. The frequently used strategy of viral absorption with added cations (Mg(2+)) and elution with acid were inefficient and more prone to inhibition, and will result in underestimation of the prevalence and concentrations of HAdVs and HPyVs markers in environmental waters

Vitamin B6 Deficiency and Galactose Induced Alterations in Morphology and Osmotic Fragility of Rat Erythrocytes

Recently, red blood cells have been investigated mainly for alterations in ion transporting capacity, membrane bound enzymes or modifications in the structure of its individual constituents in clinical and experimental urolithiasis. However, the implication of such modifications on the physical state or morphology of cells has not been investigated. Scanning electron microscopic studies performed in vitamin B6 deficient and/or galactose fed rat (established hyperoxaluric models) erythrocytes, showed the presence of large number of stomatocytes, spherocytes and other variously deformed cells as compared to discocytic cells seen in normal control group. These changes in shape were in concurrence with red cell osmotic fragility, which decreased both in vitamin B6 deficient and vitamin B6 deficient + galactose fed group (19% and 33% hemolysis at 4 g/l NaCl, respectively) while it increased in galactose control group (73% hemolysis at 4 g/l NaCl) as compared to normal control group (55 % hemolysis at 4 g/l NaCl). These morphological and physical state alterations could be correlated with red blood cells\u27 membrane cholesterol and phospholipid sub-class distribution. These findings suggest that some structural membrane changes occur due to vitamin B6 deficiency and/or galactose feeding, which may be responsible for the altered membrane functions known to be associated with pathogenesis of urolithiasis

Land Use Planning in Integrated Watershed Development Program for Improving Livelihoods

Globally, rain-fed agriculture plays an important role to achieve food security (Rockstrom et al. 2007) as 80% of the world’s agricultural land area is rainfed and generates 58% of the world’s staple foods (SIWI 2001). Most food for poor communities in the developing countries is produced in rain-fed areas for e.g. in sub-Saharan Africa (SSA) more than 95% of the farmed land is rain-fed, whereas the corresponding figure for Latin America is nearly 90%, for South Asia about 60%, for East Asia 65% and for Near East and North Africa 75%. In India, 60% of 142 million ha (Mha) arable land is rain-fed. The rain-fed areas are the hot spots of poverty, malnutrition, water scarcity, severe land degradation; and the investments in the rainfed agriculture pose serious challenges as large numbers of households are small land holders (Wani et al. 2009). These areas are also prone to more adverse impacts of climate change due to lack of technologies and necessary resource to cope with the challenges of global warming

Animal-Friendly Affinity Reagents: Replacing the Needless in the Haystack

The multibillion-dollar global antibody industry produces an indispensable resource but that is generated using millions of animals. Despite the irrefutable maturation and availability of animal-friendly affinity reagents (AFAs) employing na€ive B lymphocyte or synthetic recombinant technologies expressed by phage display, animal immunisation is still authorised for antibody production. Remarkably, replacement opportunities have been overlooked, despite the enormous potential reduction in animal use. Directive 2010/63/EU requires that animals are not used where alternatives exist. To ensure its implementation, we have engaged in discussions with the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) and the Directorate General for Environment to carve out an EU-led replacement strategy. Measures must be imposed to avoid outsourcing, regulate commercial production, and ensure that antibody producers are fully supported

Toolbox approaches using molecular markers and 16S rRNA gene amplicon data sets for identification of fecal pollution in surface water

In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small