Comparison of molecular markers to detect fresh sewage in environmental waters

Human-specific Bacteroides HF183 (HS-HF183), human-specific Enterococci faecium esp (HS-esp), human-specific adenoviruses (HS-AVs) and human-specific polyomaviruses (HS-PVs) assays were evaluated in freshwater, seawater and distilled water to detect fresh sewage. The sewage spiked water samples were also tested for the concentrations of traditional fecal indicators (i.e., Escherichia coli, enterococci and Clostridium perfringens) and enteric viruses such as enteroviruses (EVs), sapoviruses (SVs), and torquetenoviruses (TVs). The overall host-specificity of the HS-HF183 marker to differentiate between humans and other animals was 98%. However, the HS-esp, HS-AVs and HS-PVs showed 100% hostspecificity. All the human-specific markers showed >97% sensitivity to detect human fecal pollution. E. coli, enterococci and, C. perfringens were detected up to dilutions of sewage 10_5, 10_4 and 10_3 respectively.HS-esp, HS-AVs, HS-PVs, SVs and TVs were detected up to dilution of sewage 10_4 whilst EVs were detected up to dilution 10_5. The ability of the HS-HF183 marker to detect freshsewagewas3–4 orders ofmagnitude higher than that of the HS-esp and viral markers. The ability to detect fresh sewage in freshwater, seawater and distilled water matrices was similar for human-specific bacterial and viral marker. Based on our data, it appears that human-specific molecular markers are sensitive measures of fresh sewage pollution, and the HS-HF183 marker appears to be the most sensitive among these markers in terms of detecting fresh sewage. However, the presence of the HS-HF183 marker in environmental waters may not necessarily indicate the presence of enteric viruses due to their high abundance in sewage compared to enteric viruses. More research is required on the persistency of these markers in environmental water samples in relation to traditional fecal indicators and enteric pathogens.

Evaluation of multiple sewage-associated bacteroides PCR markers for sewage pollution tracking

The host specificity of the five published sewage-associated Bacteroides markers (i.e., HF183, BacHum, HuBac, BacH and Human-Bac) was evaluated in Southeast Queensland, Australia by testing fecal DNA samples (n = 186) from 11 animal species including human fecal samples collected via influent to a sewage treatment plant (STP). All human fecal samples (n = 50) were positive for all five markers indicating 100% sensitivity of these markers. The overall specificity of the HF183 markers to differentiate between humans and animals was 99%. The specificities of the BacHum and BacH markers were > 94%, suggesting that these markers are suitable for sewage pollution in environmental waters in Australia. The BacHum (i.e., 63% specificity) and Human-Bac (i.e., 79% specificity) markers performed poorly in distinguishing between the sources of human and animal fecal samples. It is recommended that the specificity of the sewage-associated markers must be rigorously tested prior to its application to identify the sources of fecal pollution in environmental waters.

Investigation of highly regulated promoters for the production of recombinant proteins in plants

Plants have been identified as promising expression systems for the commercial production of recombinant proteins. Plant-based protein production or “biofarming” offers a number of advantages over traditional expression systems in terms of scale of production, the capacity for post-translation processing, providing a product free of contaminants and cost effectiveness. A number of pharmaceutically important and commercially valuable proteins, such as antibodies, biopharmaceuticals and industrial enzymes are currently being produced in plant expression systems. However, several challenges still remain to improve recombinant protein yield with no ill effect on the host plant. The ability for transgenic plants to produce foreign proteins at commercially viable levels can be directly related to the level and cell specificity of the selected promoter driving the transgene. The accumulation of recombinant proteins may be controlled by a tissue-specific, developmentally-regulated or chemically-inducible promoter such that expression of recombinant proteins can be spatially- or temporally- controlled. The strict control of gene expression is particularly useful for proteins that are considered toxic and whose expression is likely to have a detrimental effect on plant growth. To date, the most commonly used promoter in plant biotechnology is the cauliflower mosaic virus (CaMV) 35S promoter which is used to drive strong, constitutive transgene expression in most organs of transgenic plants. Of particular interest to researchers in the Centre for Tropical Crops and Biocommodities at QUT are tissue-specific promoters for the accumulation of foreign proteins in the roots, seeds and fruit of various plant species, including tobacco, banana and sugarcane. Therefore this Masters project aimed to isolate and characterise root- and seed-specific promoters for the control of genes encoding recombinant proteins in plant-based expression systems. Additionally, the effects of matching cognate terminators with their respective gene promoters were assessed. The Arabidopsis root promoters ARSK1 and EIR1 were selected from the literature based on their reported limited root expression profiles. Both promoters were analysed using the PlantCARE database to identify putative motifs or cis-acting elements that may be associated with this activity. A number of motifs were identified in the ARSK1 promoter region including, WUN (wound-inducible), MBS (MYB binding site), Skn-1, and a RY core element (seed-specific) and in the EIR1 promoter region including, Skn-1 (seed-specific), Box-W1 (fungal elicitor), Aux-RR core (auxin response) and ABRE (ABA response). However, no previously reported root-specific cis-acting elements were observed in either promoter region. To confirm root specificity, both promoters, and truncated versions, were fused to the GUS reporter gene and the expression cassette introduced into Arabidopsis via Agrobacterium-mediated transformation. Despite the reported tissue-specific nature of these promoters, both upstream regulatory regions directed constitutive GUS expression in all transgenic plants. Further, similar levels of GUS expression from the ARSK1 promoter were directed by the control CaMV 35S promoter. The truncated version of the EIR1 promoter (1.2 Kb) showed some differences in the level of GUS expression compared to the 2.2 Kb promoter. Therefore, this suggests an enhancer element is contained in the 2.2 Kb upstream region that increases transgene expression. The Arabidopsis seed-specific genes ATS1 and ATS3 were selected from the literature based on their seed-specific expression profiles and gene expression confirmed in this study as seed-specific by RT-PCR analysis. The selected promoter regions were analysed using the PlantCARE database in order to identify any putative cis elements. The seed-specific motifs GCN4 and Skn-1 were identified in both promoter regions that are associated with elevated expression levels in the endosperm. Additionaly, the seed-specific RY element and the ABRE were located in the ATS1 promoter. Both promoters were fused to the GUS reporter gene and used to transform Arabidopsis plants. GUS expression from the putative promoters was consitutive in all transgenic Arabidopsis tissue tested. Importantly, the positive control FAE1 seed-specific promoter also directed constitutive GUS expression throughout transgenic Arabidopsis plants. The constitutive nature seen in all of the promoters used in this study was not anticipated. While variations in promoter activity can be caused by a number of influencing factors, the variation in promoter activity observed here would imply a major contributing factor common to all plant expression cassettes tested. All promoter constructs generated in this study were based on the binary vector pCAMBIA2300. This vector contains the plant selection gene (NPTII) under the transcriptional control of the duplicated CaMV 35S promoter. This CaMV 35S promoter contains two enhancer domains that confer strong, constitutive expression of the selection gene and is located immediately upstream of the promoter-GUS fusion. During the course of this project, Yoo et al. (2005) reported that transgene expression is significantly affected when the expression cassette is located on the same T-DNA as the 35S enhancer. It was concluded, the trans-acting effects of the enhancer activate and control transgene expression causing irregular expression patterns. This phenomenon seems the most plausible reason for the constitutive expression profiles observed with the root- and seed-specific promoters assessed in this study. The expression from some promoters can be influenced by their cognate terminator sequences. Therefore, the Arabidopsis ARSK1, EIR1, ATS1 and ATS3 terminator sequences were isolated and incorporated into expression cassettes containing the GUS reporter gene under the control of their cognate promoters. Again, unrestricted GUS activity was displayed throughout transgenic plants transformed with these reporter gene fusions. As previously discussed constitutive GUS expression was most likely due to the trans-acting effect of the upstream CaMV 35S promoter in the selection cassette located on the same T-DNA. The results obtained in this study make it impossible to assess the influence matching terminators with their cognate promoters have on transgene expression profiles. The obvious future direction of research continuing from this study would be to transform pBIN-based promoter-GUS fusions (ie. constructs containing no CaMV 35S promoter driving the plant selection gene) into Arabidopsis in order to determine the true tissue specificity of these promoters and evaluate the effects of their cognate 3’ terminator sequences. Further, promoter truncations based around the cis-elements identified here may assist in determining whether these motifs are in fact involved in the overall activity of the promoter.

Health protection

Our understanding of how the environment can impact human health has evolved and expanded over the centuries, with concern and interest dating back to ancient times. For example, over 4000 years ago, a civilisation in northern India tried to protect the health of its citizens by constructing and positioning buildings according to strict building laws, by having bathrooms and drains, and by having paved streets with a sewerage system (Rosen 1993). In more recent times, the ‘industrial revolution’ played a dominant role in shaping the modern world, and with it the modern public health system. This era was signified by rapid progress in technology, the growth of transportation and the expansion of the market economy, which lead to the organisation of industry into a factory system. This meant that labour had to be brought to the factories and by the 1820s, poverty and social distress (including overcrowding and infrequent sewage and garbage disposal) was more widespread than ever. These circumstances, therefore, lead to the rise of the ‘sanitary revolution’ and the birth of modern public health (Rosen 1993). The sanitary revolution has also been described as constituting the beginning of the first wave of environmental concern, which continued until after World War 2 when major advances in engineering and chemistry substantially changed the face of industry, particularly the chemical sector. The second wave of environmental concern came in the mid to late 20th century and was dominated by the environmental or ecology movement. A landmark in this era was the 1962 publication of the book Silent Spring by Rachel Carson. This identified for the first time the dramatic effects on the ecosystem of the widespread use of the organochlorine pesticide, DDT. The third wave of environmental concern commenced in the 1980s and continues today. The accelerated rate of economic development, the substantial increase in the world population and the globalisation of trade have dramatically changed the production methods and demand for goods in both developed and developing countries. This has lead to the rise of ‘sustainable development’ as a key driver in environmental planning and economic development (Yassi et al 2001). The protection of health has, therefore, been a hallmark of human history and is the cornerstone of public health practice. This chapter introduces environmental health and how it is managed in Australia, including a discussion of the key generic management tools. A number of significant environmental health issues and how they are specifically managed are then discussed, and the chapter concludes by discussing sustainable development and its links with environmental health.

Evaluating sewage associated JCV and BKV polyomaviruses for sourcing human fecal pollution in a coastal river in Southeast Queensland Australia

In this study, the host-sensitivity and -specificity of JCV and BKV polyomaviruses were evaluated by testing wastewater/fecal samples from nine host groups in Southeast Queensland, Australia. The JCV and BKV polyomaviruses were detected in 48 human wastewater samples collected from the primary and secondary effluent suggesting high sensitivity of these viruses in human wastewater. Of the 81 animal wastewater/fecal samples tested, 80 were PCR negative for this marker. Only one sample from pig wastewater was positive. Nonetheless, the overall host-specificity of these viruses to differentiate between human and animal wastewater/fecal samples was 0.99. To our knowledge, this is the first study in Australia that reports the high specificity of JCV and BKV polyomaviruses. To evaluate the field application of these viruses to detect human fecal pollution, 20 environmental samples were collected from a coastal river. Of the 20 samples tested, 15% and 70% samples exceeded the regulatory guidelines for E. coli and enterococci levels for marine waters. In all, 5 (25%) samples were PCR positive for JCV and BKV indicated the presence of human fecal pollution in the studied river. The results suggest that JCV and BKV detection using PCR could be a useful tool for the identification of human sourced fecal pollution in coastal waters.

Gene transfer to plants by diverse species of bacteria

Agrobacterium is widely considered to be the only bacterial genus capable of transferring genes to plants. When suitably modified, Agrobacterium has become the most effective vector for gene transfer in plant biotechnology1. However, the complexity of the patent landscape2 has created both real and perceived obstacles to the effective use of this technology for agricultural improvements by many public and private organizations worldwide. Here we show that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer to a number of diverse plants. These plant-associated symbiotic bacteria were made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a suitable binary vector. This alternative to Agrobacterium-mediated technology for crop improvement, in addition to affording a versatile ‘open source’ platform for plant biotechnology, may lead to new uses of natural bacteria– plant interactions to achieve plant transformation.

Analysis of alternative water sources for use in the manufacture of concrete

In Australia and many other countries worldwide, water used in the manufacture of concrete must be potable. At present, it is currently thought that concrete properties are highly influenced by the water type used and its proportion in the concrete mix, but actually there is little knowledge of the effects of different, alternative water sources used in concrete mix design. Therefore, the identification of the level and nature of contamination in available water sources and their subsequent influence on concrete properties is becoming increasingly important. Of most interest, is the recycled washout water currently used by batch plants as mixing water for concrete. Recycled washout water is the water used onsite for a variety of purposes, including washing of truck agitator bowls, wetting down of aggregate and run off. This report presents current information on the quality of concrete mixing water in terms of mandatory limits and guidelines on impurities as well as investigating the impact of recycled washout water on concrete performance. It also explores new sources of recycled water in terms of their quality and suitability for use in concrete production. The complete recycling of washout water has been considered for use in concrete mixing plants because of the great benefit in terms of reducing the cost of waste disposal cost and environmental conservation. The objective of this study was to investigate the effects of using washout water on the properties of fresh and hardened concrete. This was carried out by utilizing a 10 week sampling program from three representative sites across South East Queensland. The sample sites chosen represented a cross-section of plant recycling methods, from most effective to least effective. The washout water samples collected from each site were then analysed in accordance with Standards Association of Australia AS/NZS 5667.1 :1998. These tests revealed that, compared with tap water, the washout water was higher in alkalinity, pH, and total dissolved solids content. However, washout water with a total dissolved solids content of less than 6% could be used in the production of concrete with acceptable strength and durability. These results were then interpreted using chemometric techniques of Principal Component Analysis, SIMCA and the Multi-Criteria Decision Making methods PROMETHEE and GAIA were used to rank the samples from cleanest to unclean. It was found that even the simplest purifying processes provided water suitable for the manufacture of concrete form wash out water. These results were compared to a series of alternative water sources. The water sources included treated effluent, sea water and dam water and were subject to the same testing parameters as the reference set. Analysis of these results also found that despite having higher levels of both organic and inorganic properties, the waters complied with the parameter thresholds given in the American Standard Test Method (ASTM) C913-08. All of the alternative sources were found to be suitable sources of water for the manufacture of plain concrete.

Quantitative PCR assay of sewage-associated Bacteroides markers to assess sewage pollution in an urban lake in Dhaka, Bangladesh

This paper aimed to assess the magnitude of sewage pollution in an urban lake in Dhaka, Bangladesh by using Quantitative PCR (qPCR) of sewage-associated Bacteroides HF183 markers. PCR was also used for the quantitative detection of ruminant wastewater-associated CF128 markers along with the enumeration of traditional fecal indicator bacteria, namely, enterococci. The number of enterococci in lake water samples ranged from 1.1 x 104 to 1.9 x 105 CFU/100 ml of water. From the 20 water samples tested, 14 (70%) and 7 (35%) were PCR positive for the HF183 and CF128 markers, respectively. The numbers of the HF183 and CF128 markers in lake water samples were 3.9 x 104 to 6.3 × 107 and 9.3 x 103 to 6.3 x 105 genomic units (GU)/100 ml of water, respectively. The high numbers of enterococci and the HF183 markers indicate sewage pollution and potential health risks to those who use the lake water for non-potable purposes such as bathing and washing clothes. This is the first study that investigated the presence of microbial source tracking (MST) markers in Dhaka, Bangladesh where diarrhoeal diseases is one of the major causes of childhood mortality. The molecular assay as used in this study can provide valuable information on the extent of sewage pollution, thus facilitating the development of robust strategies to minimise potential health risks.

Sell, give away, or donate : an exploratory study of fashion clothing disposal behaviour in two countries

This study investigates the antecedents to clothing disposal methods in two countries: Scotland and Australia. Increasing volumes of textiles are disposed of in landfill sites to the detriment of the environment. Extant research has identified the influences affecting an increased rate of purchasing and the trend to keep clothing for an ever shorter time. As such, it is imperative to examine the factors that affect consumers’ choice of clothing disposal method as limited research has been undertaken in this area of socially responsible consumption. The results of a survey administered to a sample of female consumers in the two countries identify antecedents of three forms of clothing disposal methods: selling through eBay or second-hand shops, giving away to family or friends or donating to charities. Findings show differences between the countries regarding clothing disposal behaviour. Nevertheless, general recycling behaviour was found to be the strongest predictor for donating to charities in both countries.