PubMed Health⌕ Search

Biomedical subjects

N Ashbolt

Publications and source records attributed to N Ashbolt.

9 recordsLinked to original sources
PubMed2005

Hydrogen sulphide production tests and the detection of groundwater faecal contamination by septic seepage.

The 'H2S test' is being advanced for microbiological water quality testing where conventional coliform based methods are impractical or too expensive. It involves ambient temperature incubation of water samples with nutrient formulated to generate hydrogen sulphide when 'faecal' bacteria are present. Recently a WHO review identified several concerns including the limited number of comparative studies, formulation variability, and false positives and negatives. In response we have compared the H2S test's ability to detect and quantify faecal contamination in an aquifer impacted by septic tank leachfields with measurements obtained concurrently using conventional bacterial indicators, coliphages, faecal sterol biomarkers, Cryptosporidium and Giardia. Like these other analytes, H2S testing detected a contamination gradient ranging from high (septic liquid) to moderate (exfiltration zones), to background (e.g. domestic bores), corresponding to indicator removal + dilution by factors > 10(6). Presence/absence tests could not distinguish between heavily and slightly contaminated waters, whereas multi-tube testing (e.g. 10 x 10 mL arrays) did. It was concluded that while the WHO review concerns are justified, the H2S test performance shows promise in sanitary survey work, can be improved by employing an mpn approach and has potential to aid in the protection of source water and identifying contaminated groundwater.

Environmental Monitoring↗
PubMed2003

Buffer distances for on-site sewage systems in Sydney's drinking water catchments.

Pathogens and nutrients released from on-site sewage systems represent a risk to surface and ground water quality, particularly where there are sensitive receiving waters such as in drinking water catchments. Buffer zones between on-site systems and waterways are one barrier used to protect water quality. The increased time and distance they provide increases the opportunities for the effluent purification functions of the soil to occur. A risk management model is proposed to assess the efficacy of the buffer zones in Sydney's drinking water catchments. The model is the basis for the development of performance based setback distances for on-site systems from waterways, and incorporates stochastic analysis of pathogen and nutrient transport in the environment and consideration of the effluent quality variability from on-site systems. Catchment-scale integration of contaminant transport is employed to facilitate a risk assessment of on-site systems. The risk management model also allows for the impact of on-site system management and maintenance on catchment water quality to be assessed through scenario building and feedback mechanisms.

Models, Theoretical↗
PubMed2003

Centralised versus decentralised sewage systems: a comparison of pathogen and nutrient loads released into Sydney's drinking water catchments.

Data collected from centralised and decentralised sewage treatment plants throughout Sydney's drinking water catchments was used to calculate the relative catchment loads of Cryptosporidium, enteric viruses, nitrogen and phosphorus for an initial screening assessment. Loads were assessed at median and 90 percentile values for expected and worst-cases scenarios. The expected scenario in the Sydney drinking water catchments is that decentralised systems (servicing 32,800 people) provide similar total loads to centralised systems (serving 70% of the catchment population) for total phosphorus (37,090 kg x y(-1)), Cryptosporidium (10(11) oocysts x y(-1)) and enteric viruses (9.1 x 10(13) y(-1)), but higher loads of total nitrogen (237,610 vs. 136,740 kg x y(-1)). Decentralised systems, however, were predicted to have higher loads in the worst-case scenario with 620,620 kg x y(-1) TN, 82,040 kg x y(-1) TP, 7.3 x 10(13) Cryptosporidium oocysts x y(-1) and 9 x 10(15) enteric viruses per year. Greater load variability was experienced with decentralised systems, which presumably reflects less reliability in their current operation and maintenance. Overall, catchment water quality is therefore not only affected by sewage disposal methods, but also failure issues. Decentralised system disposal to land may afford a degree of mitigation that can be enhanced, if the degree of failure is reduced.

Animals↗
PubMed1998

Rapid method for fluorescent in situ ribosomal RNA labelling of Cryptosporidium parvum.

A method for fluorescence in situ hybridization (FISH) is described that requires less than 1 h duration. Oocysts were resuspended in 50% ethanol and incubated at 80 degrees C for 10 min for simultaneous fixation and permeabilization. Samples were than incubated with the oligonucleotide probe at 48 degrees C for more than 30 min. The rRNA binding specificity of the optimized protocol was confirmed. FISH was found to be valuable as a second label for oocysts presumptively identified immunofluorescently, but required more than an order of magnitude signal amplification for independent use. The number of oligonucleotide probes bound per oocyst was compared with the copy number of 18S rRNA molecules per oocyst to provide a measure of the labelling efficiency of the FISH method. Hybridization kinetics were also analysed. These data indicate that significant further increases in the brightness of FISH-labelled oocysts cannot be achieved by further optimization of the pre-treatment and hybridization conditions.

Animals↗
PubMed1998

The use of a ribosomal RNA targeted oligonucleotide probe for fluorescent labelling of viable Cryptosporidium parvum oocysts.

A fluorescence in situ hybridization (FISH) technique has been developed for the fluorescent labelling of Cryptosporidium parvum oocysts in water samples. The FISH technique employs a fluorescently labelled oligonucleotide probe (Cry1 probe) targeting a specific sequence in the 18S ribosomal RNA (rRNA) of C. parvum. Hybridization with the Cry1 probe resulted in fluorescence of sporozoites within oocysts that were capable of excystation, while oocysts that were dead prior to fixation did not fluoresce. Correlation of the FISH method with viability as measured by in vitro excystation was statistically highly significant, with a calculated correlation coefficient of 0.998. Examination of sequence data for Cryptosporidium spp. other than C. parvum suggests that the Cry1 probe is C. parvum-specific. In addition, 19 isolates of C. parvum were tested, and all fluoresced after hybridization with the Cry1 probe. Conversely, isolates of C. baileyi and C. muris were tested and found not to fluoresce after hybridization with the Cry1 probe. The fluorescence of FISH-stained oocysts was not bright enough to enable detection of oocysts in environmental water concentrates containing autofluorescent algae and mineral particles. However, in combination with immunofluorescence staining, FISH enabled species-specific detection and viability determination of C. parvum oocysts in water samples.

Animals↗
PubMed1998

Evaluation of fluorochromes for flow cytometric detection of Cryptosporidium parvum oocysts labelled by fluorescent in situ hybridization.

Oligonucleotide probes specific to Cryptosporidium parvum (CRY1) were conjugated with a range of fluorochromes. The fluorescence after in situ hybridization (FISH) labelling of oocysts and controls was assessed. The objective was to determine the most suitable conjugate for FISH labelling, followed by analysis with a 488 nm laser flow cytometer. The most promising candidate was fluorescein isothiocyanate but only when linked to the CRY1 probe via an 18-carbon spacer arm consisting of six ethylene glycol moieties. The use of the spacer increased fluorescent signals fivefold compared with an equivalent probe in which the FITC was linked directly to the 5'-amino group of the DNA.

Animals↗
PubMed1997

A simple method for evaluating Cryptosporidium-specific antibodies used in monitoring environmental water samples.

A simple method is described for the evaluation and quality control of Cryptosporidium-specific antibodies used in monitoring environmental water samples. Purified oocysts were fluorescently labelled with a test antibody at the appropriate concentration. Labelled oocysts were analysed using flow cytometry and a region was defined on a bivariate dotplot of fluorescence versus light scatter that enclosed all oocysts. Concentrates of environmental water samples that did not contain oocysts were then incubated with the test antibody and analysed using flow cytometry. The number of particles that appeared in the region defined for oocysts was recorded and was a measure of non-specific binding. The technique provides a simple, rapid and quantitative tool for both evaluating the binding specificity of test antibodies and optimizing sample staining conditions.

Animals↗
PubMed1994

Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water.

Cryptosporidium and Giardia are common causes of waterborne disease. The currently used methods of detecting these organisms in water rely on filtration capture, immunofluorescence labelling, and epifluorescence microscopy. These methods are inefficient, labour intensive, and require a highly skilled microscopist. We describe an alternative technique using flocculation concentration, followed by flow cytometry with fluorescence activated cell sorting. Environmental samples were analysed, and protozoan-like particles were sorted and collected before confirmation with epifluorescence microscopy. The technique was found to be significantly more sensitive and considerably faster than the conventional methods.

Animals↗