Paul Monis

Comparison of in vitro growth characteristics of Cryptosporidium hominis (IdA15G1) and Cryptosporidium parvum (Iowa-IIaA17G2R1 and IIaA18G3R1)

Publisher: Springer Science and Business Media LLC

Date: 30-09-2023

DOI: 10.1007/S00436-023-07979-0

Enumeration of water-borne bacteria using viability assays and flow cytometry: A comparison to culture-based techniques

Publisher: Elsevier BV

Date: 12-2003

DOI: 10.1016/S0167-7012(03)00201-X

Abstract: Maintaining optimal conditions in catchments or distribution systems relies heavily on water authorities having access to rapid and accurate water quality data, including an indication of bacteriological quality. In this study, the BacLight bacterial viability kit and carboxyfluorescein diacetate (CFDA) were coupled with flow cytometry (FCM) for rapid detection of physiologically active bacteria from raw and potable waters taken from various locations around South Australia. Results were compared to the direct viable count (DVC) and quantitative DVC (qDVC), in addition to the culture-based methods of the heterotrophic plate count (HPC) and a commercial SimPlate technique. Raw and potable water analysis revealed that DVC and culture-based techniques reported significantly fewer viable bacteria compared to the number of physiologically active bacteria detected using the rapid FCM assays, where this difference appeared to be nonlinear across different s les. Inconclusive results were obtained using qDVC as a viability assay. In particular, HPC results were 2-4 log orders of magnitude below that reported by the FCM assays for raw waters. Few bacteria in potable waters examined were culturable by HPC, even though FCM assays reported between 5.56 x 10(2) and 3.94 x 10(4) active bacteria ml(-1). These differences may be attributed to the presence of nonheterotrophic bacteria, sublethal injury or the adoption of an active but nonculturable (ABNC) state.

The importance of systematics in parasitological research

Publisher: Elsevier BV

Date: 03-1999

DOI: 10.1016/S0020-7519(98)00216-1

Abstract: The discipline of systematics plays a central role in all branches of biology. In today's technology-orientated research world, it is important to realise the continuing value of systematics, the basic tenet of which is to combine erse types of data to produce classifications that reflect the natural history of living organisms. Accurate classification systems are crucial in the field of parasitology, not only because they provide the means to identify species and strains of parasites, but also because they provide a framework around which a parasite's biology can be studied. The construction of such a classification system is often h ered by the parasite's biology, which may preclude the application of traditional techniques or concepts (such as morphological differentiation or the biological species concept) to delineate species. It is often the case that these difficulties can be overcome by the use of molecular systematic techniques. In this paper, it is proposed that a detailed understanding of the phylogeny of a group of organisms can be used as a basis to examine other aspects of their systematics. This is illustrated using the protozoan parasite Giardia intestinalis. Data gathered using the complementary techniques of allozyme electrophoresis and nucleotide sequencing have been used to infer the phylogenetic relationships of G. intestinalis isolated from various host species. The results, supported by biological data, suggest that G. intestinalis is a species-complex. As we move towards the year 2000, molecular systematics will play an increasingly important role in elucidating host-parasite relationships. However, its use as a taxonomic tool will require a general acceptance by parasitologists and the adoption of formal procedures to allow the description of new species by these methods. The aim of this approach is not to dismiss traditional methods, but to use them in combination with contemporary methods in the true spirit of the discipline of systematics.

Profiling bacterial survival through a water treatment process and subsequent distribution system

Publisher: Oxford University Press (OUP)

Date: 07-2005

DOI: 10.1111/J.1365-2672.2005.02573.X

Abstract: To profile fractions of active bacteria and of bacteria culturable with routine heterotrophic plate count (HPC) methods through a typical water treatment process and subsequent distribution system. In doing so, investigate how water treatment affects both bacterial abundance and ersity, and reveal the identities of active bacteria not detected by traditional HPC culture. Profiling active fractions was performed by flow cytometric cell sorting of either membrane-intact (BacLight kit) or enzymatically active (carboxyfluorescein diacetate, CFDA) bacteria, followed by eubacterial 16S rDNA-directed PCR and denaturing gradient gel electrophoresis (DGGE). Water treatment significantly reduced active bacterial numbers detected by the BacLight kit and CFDA assay by 2.89 and 2.81 log respectively. Bacterial ersity was also reduced from > 20 DGGE bands in the active fractions of reservoir water to only two bands in the active fractions of finished water. These two bands represented Stenotrophomonas maltophila, initially culturable by HPC, and a Burkholderia-related species. Both species maintained measurable traits of physiological activity in distribution system bulk water but were undetected by HPC. Flow cytometric cell sorting with PCR-DGGE, to assess water treatment efficacy, identified active bacteria from a variety of major phylogenetic groups undetected by routine HPC. Following treatment S. maltophila and a Burkholderia-related species retained activity and entered distribution undetected by HPC. Methods used here demonstrate how water treatment operators can better monitor water treatment plant efficacy and assess distribution system instability by the detection and identification of active bacteria recalcitrant to routine HPC culture.

Understanding human infectious Cryptosporidium risk in drinking water supply catchments

Publisher: Elsevier BV

Date: 07-2018

DOI: 10.1016/J.WATRES.2018.03.063

Abstract: Treating drinking water appropriately depends, in part, on the robustness of source water quality risk assessments, however quantifying the proportion of infectious, human pathogenic Cryptosporidium oocysts remains a significant challenge. We analysed 962 source water s les across nine locations to profile the occurrence, rate and timing of infectious, human pathogenic Cryptosporidium in surface waters entering drinking water reservoirs during rainfall-runoff conditions. At the catchment level, average infectivity over the four-year study period reached 18% however, most locations averaged <5%. The maximum recorded infectivity fraction within a single rainfall runoff event was 65.4%, and was dominated by C. parvum. Twenty-two Cryptosporidium species and genotypes were identified using PCR-based molecular techniques the most common being C. parvum, detected in 23% of water s les. Associations between landuse and livestock stocking characteristics with Cryptosporidium were determined using a linear mixed-effects model. The concentration of pathogens in water were significantly influenced by flow and dominance of land-use by commercial grazing properties (as opposed to lifestyle properties) in the catchment (p < 0.01). Inclusion of measured infectivity and human pathogenicity data into a quantitative microbial risk assessment (QMRA) could reduce the source water treatment requirements by up to 2.67 log removal values, depending on the catchment, and demonstrated the potential benefit of collating such data for QMRAs.

Epidemiological evaluation of sewage surveillance as a tool to detect the presence of COVID-19 cases in a low case load setting

Publisher: Elsevier BV

Date: 09-2021

DOI: 10.1016/J.SCITOTENV.2021.147469

Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria

Publisher: Oxford University Press (OUP)

Date: 05-2008

DOI: 10.1111/J.1365-2672.2007.03676.X

Abstract: To develop and test a real-time PCR assay to detect and quantify genes specific to Cylindrospermopsis sp. and cylindrospermopsin-producing cyanobacteria. A duplex real-time PCR assay was developed that targets a cylindrospermopsin-specific and Cylindrospermopsis raciborskii-specific DNA sequence. The C. raciborskii-specific sequence was based on the rpoC1 DNA-dependent RNA polymerase gene, whilst the cylindrospermopsin-specific sequence was selected by surveying an extensive number of potential cylindrospermopsin-producing cyanobacterial strains for genes implicated in toxin production, aoaA, aoaB and aoaC. In toxic strains, sequences of each of these three genes were always present whilst in nontoxic strains the distribution of these sequences was patchy, resulting in what are likely to be natural deletion mutants. The real-time assay was optimized on a fixed and portable device, with results indicating that the reliable limit of detection for the assay was 100 copies per reaction or 1000 cells ml(-1) for both target sequences on both devices. In routine environmental s les enumerated by microscopy, the assay results were positive for all s les where C. raciborskii cells were observed at >1000 cells ml(-1) and negative in 15 s les where no C. raciborskii cells were observed. In field s les, the number of copies of the rpoC1 sequence more closely approximated the number of cells enumerated by microscopy, the number of copies of the pks sequence and detection of the toxin-specific sequence matched the results of toxin testing. The duplex real-time PCR assay was a sensitive and rapid method for detecting potential cylindrospermopsin-producing cyanobacteria in the laboratory or in the field. The observation of probable natural deletion mutants provides further evidence that the aoaA, aoaB and aoaC genes are involved in toxin production. This assay provides a new monitoring capability for tracking cylindrospermopsin-producing cyanobacteria that are an emerging threat to water quality.

Novel toxic effects associated with a tropical Limnothrix/Geitlerinema‐like cyanobacterium

Publisher: Wiley

Date: 05-05-2011

DOI: 10.1002/TOX.20552

Abstract: The presence of a toxic strain of a fine filamentous cyanobacterium belonging to the Oscillatorialean family Pseudanabaenacea was detected during a survey of cyanobacterial taxa associated with the presence of cylindrospermopsin in dams in Central Queensland (Australia). The strain, AC0243, was isolated and cultured, its genomic DNA extracted and 16S RNA gene sequenced. Phylogenetic analysis placed AC0243 with Limnothrix species, although this genus appears polyphyletic. Moreover, not all morphological characters are consistent with this genus but more closely fit the description of Geitlerinema unigranulatum (R.N. Singh) Komárek and Azevedo. The potential toxic effects of AC0243 extract were assessed chemically and biologically. Cell free protein synthesis was inhibited by the extract. Exposure of Vero cells to the extract resulted in a significant reduction in cellular ATP levels following 24-72 h incubation. The presence of cylindrospermopsin was excluded based on the nature of responses obtained in cell and cell-free assays in addition, (i) it could not be detected by HPLC, LC-MS, or immunological assay, and (ii) no genes currently associated with the production of cylindrospermopsin were found in the genome. Other known cyanobacterial toxins were not detected. The apparent novelty of this toxin is discussed.

Cryptosporidium and Giardia-zoonoses: Fact or fiction?

Publisher: Elsevier BV

Date: 11-2003

DOI: 10.1016/J.MEEGID.2003.08.003

Abstract: Giardia and Cryptosporidium are enteric protozoan parasites that infect a wide range of vertebrate hosts. Both are transmitted either by direct faecal/oral contact or by the ingestion of contaminated food or water. The discovery of morphologically similar organisms infecting humans and a variety of mammals and birds has led to the proposal that both Cryptosporidium and Giardia are zoonotic (i.e. transmitted in nature between humans and animals). Transmission between humans and animals has been supported by cross-infection studies. However, closer examination of many of these studies reveals limitations in the methodologies utilised. More recent molecular genetic studies have demonstrated considerable genetic ersity among isolates of the same species of Giardia and Cryptosporidium, suggesting that these species are in fact species complexes and that some of these novel species may be host-specific. This paper will critically examine the evidence for the zoonotic transmission of these parasites.

Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis

Publisher: Elsevier BV

Date: 05-2005

DOI: 10.1016/J.AB.2005.01.046

Abstract: Following the initial report of the use of SYBR Green I for real-time polymerase chain reaction (PCR) in 1997, little attention has been given to the development of alternative intercalating dyes for this application. This is surprising considering the reported limitations of SYBR Green I, which include limited dye stability, dye-dependent PCR inhibition, and selective detection of licons during DNA melting curve analysis of multiplex PCRs. We have tested an alternative to SYBR Green I and report the first detailed evaluation of the intercalating dye SYTO9. Our findings demonstrate that SYTO9 produces highly reproducible DNA melting curves over a broader range of dye concentrations than does SYBR Green I, is far less inhibitory to PCR than SYBR Green I, and does not appear to selectively detect particular licons. The low inhibition and high melting curve reproducibility of SYTO9 means that it can be readily incorporated into a conventional PCR at a broad range of concentrations, allowing closed tube analysis by DNA melting curve analysis. These features simplify the use of intercalating dyes in real-time PCR and the improved reproducibility of DNA melting curve analysis will make SYTO9 useful in a diagnostic context.

Nucleic acid amplification-based techniques for pathogen detection and identification

Publisher: Elsevier BV

Date: 2006

DOI: 10.1016/J.MEEGID.2005.08.004

Environmental temperature controls Cryptosporidium oocyst metabolic rate and associated retention of infectivity

Publisher: American Society for Microbiology

Date: 07-2005

DOI: 10.1128/AEM.71.7.3848-3857.2005

Abstract: Cryptosporidium is a significant cause of water-borne enteric disease throughout the world and represents a challenge to the water industry and a threat to public health. In this study we report the use of a cell culture-TaqMan PCR assay to measure oocyst inactivation rates in reagent-grade and environmental waters over a range of temperatures. While oocysts incubated at 4°C and 15°C remained infective over the 12-week holding period, we observed a 4 log 10 reduction in infectivity for both 20 and 25°C incubation treatments at 12 and 8 weeks, respectively, for all water types examined, a faster rate of inactivation for oocysts than previously reported. This temperature-dependent inactivation was further investigated using a simple and rapid ATP assay described herein. Time course experiments performed in reagent-grade water at incubation temperatures of 4, 15, 20, 25, 30, and 37°C identified a close relationship between oocyst infectivity and oocyst ATP content, demonstrating that temperature inactivation at higher temperatures is a function of increased oocyst metabolic activity. While water quality did not affect oocyst inactivation, biological antagonism appears to be a key factor affecting oocyst removal from environmental waters. Both the cell culture-TaqMan PCR assay and the ATP assay provide a sensitive and quantitative method for the determination of environmental oocyst inactivation, providing an alternative to the more costly and time-consuming mouse infection assay. The findings presented here relating temperature to oocyst inactivation provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in water.

Wastewater monitoring for SARS-CoV-2

Publisher: CSIRO Publishing

Date: 2021

DOI: 10.1071/MA21006

Abstract: Wastewater monitoring (WM) of SARS-CoV-2 from sewers was applied throughout the world early in the COVID-19 pandemic. Sharing of protocols and experiences in WM of SARS-CoV-2 by national and international researchers and practitioners has been vital to ensuring the sensitivity and specificity of the methods. WM has been a valuable adjunct to human clinical testing, and when positive results occur in sewage, community testing has been increased. WM findings allow public health officials to track and respond to the impacts of loosening lockdown restrictions, demonstrating when return to normal social activities might occur without a resurgence of rapid community transmission, and they are particularly useful in areas with low human case numbers and/or low clinical testing rates. New research is required to address several practical knowledge gaps, for ex le, s ling protocols, prediction of case prevalence from viral numbers by modelling, and determination of detection limits. Communication to the Australian public of WM of SARS-CoV-2 has been via interactive, visual dashboards. Once SARS-CoV-2 vaccinations are introduced, WM could help track the underlying circulation of the virus in the population, the spread of known variants and its future evolution.

Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples

Publisher: Elsevier BV

Date: 12-2014

DOI: 10.1016/J.IJPARA.2014.08.004

Abstract: Clinical microbiology laboratories rely on quantitative PCR for its speed, sensitivity, specificity and ease-of-use. However, quantitative PCR quantitation requires the use of a standard curve or normalisation to reference genes. Droplet digital PCR provides absolute quantitation without the need for calibration curves. A comparison between droplet digital PCR and quantitative PCR-based analyses was conducted for the enteric parasite Cryptosporidium, which is an important cause of gastritis in both humans and animals. Two loci were analysed (18S rRNA and actin) using a range of Cryptosporidium DNA templates, including recombinant plasmids, purified haemocytometer-counted oocysts, commercial flow cytometry-counted oocysts and faecal DNA s les from sheep, cattle and humans. Each method was evaluated for linearity, precision, limit of detection and cost. Across the same range of detection, both methods showed a high degree of linearity and positive correlation for standards (R(2)⩾0.999) and faecal s les (R(2)⩾0.9750). The precision of droplet digital PCR, as measured by mean Relative Standard Deviation (RSD %), was consistently better compared with quantitative PCR, particularly for the 18S rRNA locus, but was poorer as DNA concentration decreased. The quantitative detection of quantitative PCR was unaffected by DNA concentration, but droplet digital PCR quantitative PCR was less affected by the presence of inhibitors, compared with quantitative PCR. For most templates analysed including Cryptosporidium-positive faecal DNA, the template copy numbers, as determined by droplet digital PCR, were consistently lower than by quantitative PCR. However, the quantitations obtained by quantitative PCR are dependent on the accuracy of the standard curve and when the quantitative PCR data were corrected for pipetting and DNA losses (as determined by droplet digital PCR), then the sensitivity of both methods was comparable. A cost analysis based on 96 s les revealed that the overall cost (consumables and labour) of droplet digital PCR was two times higher than quantitative PCR. Using droplet digital PCR to precisely quantify standard dilutions used for high-throughput and cost-effective lifications by quantitative PCR would be one way to combine the advantages of the two technologies.

Monitoring of geosmin producing Anabaena circinalis using quantitative PCR

Publisher: Elsevier BV

Date: 02-2014

DOI: 10.1016/J.WATRES.2013.10.028

Abstract: Geosmin is one of the most commonly detected off-flavor chemicals present in reservoirs and drinking water systems. Quantitative real-time PCR (qPCR) is useful for quantifying geosmin-producers by focusing on the gene encoding geosmin synthase, which is responsible for geosmin synthesis. In this study, several primers and probes were designed and evaluated to detect the geosmin synthase gene in cyanobacteria. The specificity of primer and probe sets was tested using 21 strains of laboratory cultured cyanobacteria isolated from surface waters in Australia (18) and Taiwan (2), including 6 strains with geosmin producing ability. The results showed that the primers designed in this study could successfully detect all geosmin producing strains tested. The selected primers were used in a qPCR assay, and the calibration curves were linear from 5 × 10(1) to 5 × 10(5) copies mL(-1), with a high correlation coefficient (R(2) = 0.999). This method was then applied to analyze s les taken from Myponga Reservoir, South Australia, during a cyanobacterial bloom event. The results showed good correlations between qPCR techniques and traditional methods, including cell counts determined by microscopy and geosmin concentration measured using gas chromatography (GC) coupled with a mass selective detector (MSD). Results demonstrate that qPCR could be used for tracking geosmin-producing cyanobacteria in drinking water reservoirs. The qPCR assay may provide water utilities with the ability to properly characterize a taste and odor episode and choose appropriate management and treatment options.

Virus removal of new and aged UF membranes at full-scale in a wastewater reclamation plant

Publisher: Royal Society of Chemistry (RSC)

Date: 2016

DOI: 10.1039/C6EW00197A

Abstract: We examined the consequence of long-term membrane ageing on the virus rejection performance of full-scale ultrafiltration membranes to better understand health risks associated with membrane use in wastewater reuse schemes.

Variation in Giardia: Implications for taxonomy and epidemiology

Publisher: Elsevier

Date: 2004

DOI: 10.1016/S0065-308X(04)58002-8

Inactivation, removal, and regrowth potential of opportunistic pathogens and antimicrobial resistance genes in recycled water systems

Publisher: Elsevier BV

Date: 08-2021

DOI: 10.1016/J.WATRES.2021.117324

Novel lineages of Giardia intestinalis identified by genetic analysis of organisms isolated from dogs in Australia

Publisher: Cambridge University Press (CUP)

Date: 1998

DOI: 10.1017/S0031182097002011

Abstract: Infection of suckling mice with Giardia trophozoites recovered from the intestines of 11 dogs autopsied in Central and Southern Australia in each case produced an established isolate. In contrast, only 1 isolate was obtained by inoculation of faecal cysts. The organisms grew poorly in comparison with isolates from humans or non-canine animal hosts. Light microscopy revealed that the trophozoites had median bodies with the ‘claw hammer’ appearance typical of G. intestinalis (syn. G. duodenalis , G. lamblia ) but that they differed in shape and nuclear morphology from axenic isolates of human or canine origin. Allozymic analysis of electrophoretic data representing 26 loci and phylogenetic analysis of nucleotide sequences obtained from DNA lified from the glutamate dehydrogenase locus showed that the 11 isolates examined from Australian dogs were genetically distinct from all isolates of G. intestinalis that have been established previously from humans and animals, and also from G. muris . Both analytical methods placed 10 of the Australian canine isolates into a unique genetic lineage (designated Assemblage C) and the eleventh into a deep-rooted second branch (designated Assemblage D), each well separated from the 2 lineages (Assemblages A and B) of G. intestinalis that encompass all the genotypes known to infect humans. In contrast, 4 axenic isolates derived from dogs in Canada and Europe (the only other isolates to have been established from dogs) have genotypes characteristic of genetic Assemblages A or B. The findings indicate that the novel Giardia identified in these rural Australian dogs have a restricted host range, possibly confined to canine species. The poor success rate in establishing Giardia from dogs in vitro suggests, further, that similar genotypes may predominate as canine parasites world-wide. The absence of such organisms among isolates of Giardia that have been established from humans by propagation in suckling mice indicates that they are unlikely to infect humans. However, infection of humans by those dog-derived genotypes that grow in vitro cannot be excluded.

Flow cytometric assessment of distinct physiological stages within Cryptosporidium parvum sporozoites post-excystation

Publisher: Cambridge University Press (CUP)

Date: 24-06-2009

DOI: 10.1017/S0031182009006519

Abstract: Cryptosporidium parvum are protozoan parasites responsible for outbreaks of gastrointestinal disease worldwide. Within the apical complex of this organism reside numerous vesicular secretory organelles and their discharge has been identified as essential for sporozoite motility, cell attachment and penetration. Traditionally, investigation of apical organelle discharge has relied on microscopic and immunochemical hybridization techniques. In this study we demonstrate for the first time how flow cytometry, in combination with vital dye staining, provides an avenue for discrimination of distinct physiological events occurring within Cryptosporidium sporozoites post-excystation. Time-course studies of freshly excysted sporozoites were carried out at 37°C in cell-free medium, stained with the fluorescent dyes SYTO9/PI, DiBAC 4 (3), Fluo-4 AM or FM1-43 and analysed by flow cytometry. Significant decreases in sporozoite plasma membrane permeability and increased membrane depolarization were found to be accompanied by concomitant increases in intracellular calcium. Subsequent to these changes, large increases in exocytosed vesicular membrane were apparent. In addition, by measuring side and forward angle light scatter we were able to assess changes in internal granularity and size of sporozoites post-excystation. These observations were suggestive of rapid mobilization, utilization and discharge of apical organelles within sporozoites, which we relate to changes in sporozoite infectivity, ATP levels and total secreted soluble protein.

It's official – Cryptosporidium is a gregarine: What are the implications for the water industry?

Publisher: Elsevier BV

Date: 11-2016

DOI: 10.1016/J.WATRES.2016.09.013

Abstract: Parasites of the genus Cryptosporidium are a major cause of diarrhoea and ill-health in humans and animals and are frequent causes of waterborne outbreaks. Until recently, it was thought that Cryptosporidium was an obligate intracellular parasite that only replicated within a suitable host, and that faecally shed oocysts could survive in the environment but could not multiply. In light of extensive biological and molecular data, including the ability of Cryptosporidium to complete its life cycle in the absence of a host and the production of novel extracellular stages, Cryptosporidium has been formally transferred from the Coccidia, to a new subclass, Cryptogregaria, with gregarine parasites. In this review, we discuss the close relationship between Cryptosporidium and gregarines and discuss the implications for the water industry.

Removal of pathogens by functionalised self-assembled monolayers

Publisher: IWA Publishing

Date: 03-2008

DOI: 10.2166/AQUA.2008.034

Cryptosporidiumcell culture infectivity assay design

Publisher: Cambridge University Press (CUP)

Date: 18-03-2011

DOI: 10.1017/S0031182011000217

Abstract: Members of the genus Cryptosporidium , which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture, in vitro growth has been h ered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design of in vitro culture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.

Extracellular excystation and development of Cryptosporidium: tracing the fate of oocysts within Pseudomonas aquatic biofilm systems

Publisher: Springer Science and Business Media LLC

Date: 18-11-2014

DOI: 10.1186/S12866-014-0281-8

Molecular epidemiology: assumptions and limitations of commonly applied methods

Publisher: Elsevier BV

Date: 06-1998

DOI: 10.1016/S0020-7519(98)00042-3

Abstract: An understanding of the epidemiology of a disease (i.e. its aetiology, transmission patterns) is crucial for the development and implementation of effective management practices. This requires sound epidemiological data. It is therefore important that scientists understand the assumptions and limitations of the methods used to gather such data. The aim of this paper is to discuss some of the assumptions and limitations of PCR-based methods used in studies of epidemiology. Since its development, PCR has had a major impact in the biological sciences. The ability to selectively lify a specific region of the genome from a small amount of DNA makes this technique particularly useful as a diagnostic tool. A variety of PCR-based methods are available which can be used to identify strains and species of parasites. Some of these methods, such as random lification of polymorphic DNA, have intrinsic properties which can limit their application. Other methods, such as PCR-restriction fragment length polymorphism, require the availability of a sound taxonomic or genetic framework for the development of any diagnostic system for a particular organism. The problems encountered developing diagnostic probes in the absence of such a framework will be discussed using Giardia intestinalis as an ex le.

Comparison of drinking water treatment process streams for optimal bacteriological water quality

Publisher: Elsevier BV

Date: 08-2012

DOI: 10.1016/J.WATRES.2012.04.041

Abstract: Four pilot-scale treatment process streams (Stream 1 - Conventional treatment (coagulation/flocculation/dual media filtration) Stream 2 - Magnetic ion exchange (MIEX)/Conventional treatment Stream 3 - MIEX/Conventional treatment/granular activated carbon (GAC) filtration Stream 4 - Microfiltration/nanofiltration) were commissioned to compare their effectiveness in producing high quality potable water prior to disinfection. Despite receiving highly variable source water quality throughout the investigation, each stream consistently reduced colour and turbidity to below Australian Drinking Water Guideline levels, with the exception of Stream 1 which was difficult to manage due to the reactive nature of coagulation control. Of particular interest was the bacteriological quality of the treated waters where flow cytometry was shown to be the superior monitoring tool in comparison to the traditional heterotrophic plate count method. Based on removal of total and active bacteria, the treatment process streams were ranked in the order: Stream 4 (average log removal of 2.7) > Stream 2 (average log removal of 2.3) > Stream 3 (average log removal of 1.5) > Stream 1 (average log removal of 1.0). The lower removals in Stream 3 were attributed to bacteria detaching from the GAC filter. Bacterial community analysis revealed that the treatments affected the bacteria present, with the communities in streams incorporating conventional treatment clustering with each other, while the community composition of Stream 4 was very different to those of Streams 1, 2 and 3. MIEX treatment was shown to enhance removal of bacteria due to more efficient flocculation which was validated through the novel application of the photometric dispersion analyser.

Independent validation and regulatory agency approval for high rate algal ponds to treat wastewater from rural communities

Publisher: Royal Society of Chemistry (RSC)

Date: 2018

DOI: 10.1039/C7EW00228A

Abstract: This is the first validation of a HRAP accepted by a regulatory agency and resulted in the system being incorporated into the South Australian Community Wastewater Management Scheme – as depicted.

Field based pilot-scale drinking water distribution system: Simulation of long hydraulic retention times and microbiological mediated monochloramine decay

Publisher: Elsevier BV

Date: 2018

DOI: 10.1016/J.MEX.2018.06.015

Biodegradation of geosmin by a novel Gram-negative bacterium; isolation, phylogenetic characterisation and degradation rate determination

Publisher: Elsevier BV

Date: 06-2009

DOI: 10.1016/J.WATRES.2009.04.005

Abstract: Biologically active sand filters within water treatment plants (WTPs) are now recognised as an effective barrier for the removal of geosmin. However, little is known regarding the actual microbiological processes occurring or the bacteria capable of degrading geosmin. This study reports the enrichment and isolation of a Gram-negative bacterium, Geo48, from the biofilm of a WTP sand filter where the isolate was shown to effectively degrade geosmin in idually. Experiments revealed that Geo48 degraded geosmin in a planktonic state by a pseudo-first-order mechanism. Initial geosmin concentrations ranging from 100 to 1000ng/l were shown to directly influence geosmin degradation in reservoir water by Geo48, with rate constants increasing from 0.010h(-1) (R(2)=0.93) to 0.029h(-1) (R(2)=0.97) respectively. Water temperature also influenced degradation of geosmin by Geo48 where temperatures of 11, 22 and 30 degrees C resulted in rate constants of 0.017h(-1) (R(2)=0.98), 0.023h(-1) (R(2)=0.91) and 0.019h(-1) (R(2)=0.85) respectively. Phylogenetic analysis using the 16S rRNA gene of Geo48 revealed it was a member of the Alphaproteobacteria and clustered with 99% bootstrap support with an isolate designated Geo24, a Sphingopyxis sp. previously described as degrading geosmin but only as a member of a bacterial consortium. Of the previously described bacteria, Geo48 was most similar to Sphingopyxis alaskensis (97.2% sequence similarity to a 1454bp fragment of the 16S rRNA gene). To date, this is the only study to report the isolation and characterisation of a Gram-negative bacterium from a biologically active sand filter capable of the sole degradation of geosmin.

Molecular biology techniques in parasite ecology

Publisher: Elsevier BV

Date: 05-2002

DOI: 10.1016/S0020-7519(01)00352-6

Abstract: Molecular techniques are increasingly being used to study the ecology of a variety of organisms. These techniques represent important tools for the study of the systematics, population genetics, biogeography and ecology of parasites. Here, we review the techniques that have been employed to study the ecology and systematics of parasites (including bacteria and viruses). Particular emphasis is placed on the techniques of isoenzyme electrophoresis, in situ hybridisation and nucleic acid lification to characterise parasite/microbial communities. The application of these techniques will be exemplified using ticks, bacterial endosymbionts and parasitic protozoa.

DNA‐Mediated Assembly of Gold Nanoparticles and Applications in Bioanalysis

Publisher: Wiley

Date: 23-08-2017

DOI: 10.1002/CNMA.201700177

Giardia—From Genome to Proteome

Publisher: Elsevier

Date: 2012

DOI: 10.1016/B978-0-12-394303-3.00003-7

Profiling the diversity of Cryptosporidium species and genotypes in wastewater treatment plants in Australia using next generation sequencing

Publisher: Elsevier BV

Date: 12-2018

DOI: 10.1016/J.SCITOTENV.2018.07.024

Abstract: Wastewater recycling is an increasingly popular option in worldwide to reduce pressure on water supplies due to population growth and climate change. Cryptosporidium spp. are among the most common parasites found in wastewater and understanding the prevalence of human-infectious species is essential for accurate quantitative microbial risk assessment (QMRA) and cost-effective management of wastewater. The present study conducted next generation sequencing (NGS) to determine the prevalence and ersity of Cryptosporidium species in 730 raw influent s les from 25 Australian wastewater treatment plants (WWTPs) across three states: New South Wales (NSW), Queensland (QLD) and Western Australia (WA), between 2014 and 2015. All s les were screened for the presence of Cryptosporidium at the 18S rRNA (18S) locus using quantitative PCR (qPCR), oocyst numbers were determined directly from the qPCR data using DNA standards calibrated by droplet digital PCR, and positives were characterized using NGS of 18S licons. Positives were also screened using C. parvum and C. hominis specific qPCRs. The overall Cryptosporidium prevalence was 11.4% (83/730): 14.3% (3/21) in NSW 10.8% (51/470) in QLD and 12.1% (29/239) in WA. A total of 17 Cryptosporidium species and six genotypes were detected by NGS. In NSW, C. hominis and Cryptosporidium rat genotype III were the most prevalent species (9.5% each). In QLD, C. galli, C. muris and C. parvum were the three most prevalent species (7.7%, 5.7%, and 4.5%, respectively), while in WA, C. meleagridis was the most prevalent species (6.3%). The oocyst load/Litre ranged from 70 to 18,055 oocysts/L (overall mean of 3426 oocysts/L: 4746 oocysts/L in NSW 3578 oocysts/L in QLD and 3292 oocysts/L in WA). NGS-based profiling demonstrated that Cryptosporidium is prevalent in the raw influent across Australia and revealed a large ersity of Cryptosporidium species and genotypes, which indicates the potential contribution of livestock, wildlife and birds to wastewater contamination.

Investigating source water Cryptosporidium concentration, species and infectivity rates during rainfall-runoff in a multi-use catchment

Publisher: American Society of Agricultural and Biological Engineers

Date: 03-11-2014

DOI: 10.13031/WTCW.2014-041

Biodegradation of multiple cyanobacterial metabolites in drinking water supplies

Publisher: Elsevier BV

Date: 06-2012

DOI: 10.1016/J.CHEMOSPHERE.2012.02.020

Abstract: The fate of multiple cyanobacterial metabolites was assessed in two Australian source waters. The saxitoxins were the only metabolites shown to be non-biodegradable in Myponga Reservoir water, while microcystin-LR (MCLR) and geosmin were biodegradable in this water source. Likewise, cylindrospermopsin (CYN) was shown to be biodegradable in River Murray water. The order of ease of biodegradability followed the trend: MCLR>CYN>geosmin>saxitoxins. Biodegradation of the metabolites was affected by temperature and seasonal variations with more rapid degradation at 24°C and during autumn compared with 14°C and during winter. A microcystin-degrading bacterium was isolated and shown to degrade four microcystin variants within 4 h. This bacterium, designated as TT25, was shown to be 99% similar to a Sphingopyxis sp. based on a 16S rRNA gene fragment. Isolate TT25 was shown to contain a homologue of the mlrA gene the sequence of which was 99% similar to that of a previously reported microcystin-degrader. Furthermore, isolate TT25 could degrade the microcystins in the presence of copper sulphate (0.5 mg L(-1) as Cu(2+)) which is advantageous for water authorities dosing such algicides into water bodies to control cyanobacterial blooms.

Isolation and characterization of the gene associated with geosmin production in cyanobacteria

Publisher: American Chemical Society (ACS)

Date: 30-09-2008

DOI: 10.1021/ES801465W

Multiplication of the waterborne pathogen Cryptosporidium parvum in an aquatic biofilm system

Publisher: Springer Science and Business Media LLC

Date: 19-09-2013

DOI: 10.1186/1756-3305-6-270

Cryptosporidium and the Environment-Overview and Summary

Publisher: Elsevier

Date: 2003

DOI: 10.1016/B978-044451351-9/50061-6

The isolation and microbial community analysis of hydrogen producing bacteria from activated sludge

Publisher: Oxford University Press (OUP)

Date: 19-06-2007

DOI: 10.1111/J.1365-2672.2007.03370.X

Abstract: To profile the fractions of bacteria in heat-treated activated sludge capable of producing hydrogen and subsequently to isolate those organisms and confirm their ability to produce hydrogen. Profiling the community composition of the microflora in activated sludge using 16S rRNA gene-directed polymerase chain reaction-denaturing gradient gel electrophoresis suggested that a majority of bacteria were various Clostridium species. This was confirmed by clone library analysis, where 80% of the cloned inserts were Clostridium sp. A total of five isolates were established on solid media. Three of them, designated as W1, W4 and W5, harboured the hydrogenase gene as determined by PCR and DNA sequence analysis (99% similarity). These isolates were similar to Clostridium butyricum and Clostridium diolis as determined by 16S rRNA gene sequence. A maximum hydrogen production yield of 220 ml H(2) g(-1) glucose was achieved by W5, which was grown on improved mineral medium by batch fermentation without pH adjustment and nitrogen sparging during fermentation. Accumulation of malic acid and fumaric acid during hydrogen fermentation might lead to higher hydrogen yields for W4 and W5. W1 is the first reported Clostridium species that can tolerate microaerobic conditions for producing hydrogen. Clostridium species in heat-treated activated sludge were the most commonly identified bacteria responsible for hydrogen production. Specific genetic markers for strains W1, W4 and W5 would be of great utility in investigating hydrogen production at the molecular level. Two previously described primer sets targeting hydrogenase genes were shown not to be specific, lifying other genes from nonhydrogen producers. Clostridium species isolated from heat-treated activated sludge were confirmed as hydrogen producers during dark hydrogen fermentation. The isolates will be useful for studying hydrogen production from wastewater, including the process of gene regulation and hydrogenase activity.

Cooperative biodegradation of geosmin by a consortium comprising three gram-negative bacteria isolated from the biofilm of a sand filter column

Publisher: Oxford University Press (OUP)

Date: 10-2006

DOI: 10.1111/J.1472-765X.2006.01974.X

Cryptosporidium species and subtypes in animals inhabiting drinking water catchments in three states across Australia

Publisher: Elsevier BV

Date: 05-2018

DOI: 10.1016/J.WATRES.2018.02.005

Abstract: As part of long-term monitoring of Cryptosporidium in water catchments serving Western Australia, New South Wales (Sydney) and Queensland, Australia, we characterised Cryptosporidium in a total of 5774 faecal s les from 17 known host species and 7 unknown bird s les, in 11 water catchment areas over a period of 30 months (July 2013 to December 2015). All s les were initially screened for Cryptosporidium spp. at the 18S rRNA locus using a quantitative PCR (qPCR). Positives s les were then typed by sequence analysis of an 825 bp fragment of the 18S gene and subtyped at the glycoprotein 60 (gp60) locus (832 bp). The overall prevalence of Cryptosporidium across the various hosts s led was 18.3% (1054/5774 95% CI, 17.3-19.3). Of these, 873 s les produced clean Sanger sequencing chromatograms, and the remaining 181 s les, which initially produced chromatograms suggesting the presence of multiple different sequences, were re-analysed by Next- Generation Sequencing (NGS) to resolve the presence of Cryptosporidium and the species composition of potential mixed infections. The overall prevalence of confirmed mixed infection was 1.7% (98/5774), and in the remaining 83 s les, NGS only detected one species of Cryptosporidium. Of the 17 Cryptosporidium species and four genotypes detected (Sanger sequencing combined with NGS), 13 are capable of infecting humans C. parvum, C. hominis, C. ubiquitum, C. cuniculus, C. meleagridis, C. canis, C. felis, C. muris, C. suis, C. scrofarum, C. bovis, C. erinacei and C. fayeri. Oocyst numbers per gram of faeces (g

A genetic and metabolic approach to redirection of biochemical pathways of Clostridium butyricum for enhancing hydrogen production

Publisher: Wiley

Date: 18-07-2013

DOI: 10.1002/BIT.24596

Abstract: Clostridium butyricum, a well known H(2) producing bacterium, produces lactate, butyrate, acetate, ethanol, and CO(2) as its main by-products from glucose. The conversion of pyruvate to lactate, butyrate and ethanol involves oxidation of NADH. It was hypothesized that the NADH could be increased if the formation of these by-products could be eliminated, resulting in enhancing H(2) yield. Herein, this study aimed to establish a genetic and metabolic approach for enhancing H(2) yield via redirection of metabolic pathways of a C. butyricum strain. The ethanol formation pathway was blocked by disruption of aad (encoding aldehyde-alcohol dehydrogenase) using a ClosTron plasmid. Although elimination of ethanol formation alone did not increase hydrogen production, the resulting aad-deficient mutant showed approximately 20% enhanced performance in hydrogen production with the addition of sodium acetate. This work demonstrated the possibility of improving hydrogen yield by eliminating the unfavorable by-products ethanol and lactate.

Zoonotic Cryptosporidium species in animals inhabiting sydney water catchments

Publisher: Public Library of Science (PLoS)

Date: 14-12-2016

DOI: 10.1371/JOURNAL.PONE.0168169

Solar UV reduces Cryptosporidium parvum oocyst infectivity in environmental waters

Publisher: Oxford University Press (OUP)

Date: 05-2008

DOI: 10.1111/J.1365-2672.2007.03658.X

Abstract: To determine the effect of solar radiation on Cryptosporidium parvum in tap and environmental waters. Outdoor tank experiments and a cell culture infectivity assay were used to measure solar inactivation of C. parvum oocysts in different waters. Experiments conducted on days with different levels of solar insolation identified rapid inactivation of oocysts in tap water (up to 90% inactivation within the first hour). Increased dissolved organic carbon content in environmental waters decreased solar inactivation. The role of solar ultraviolet (UV) in inactivation was confirmed by long-pass filter experiments, where UV-B was identified as the most germicidal wavelength. Reductions in oocyst infectivity following solar radiation were not related to a loss of excystation capacity. Solar UV can rapidly inactivate C. parvum in environmental waters. This is the first study to assess natural sunlight inactivation of C. parvum oocysts in surface waters and drinking water using an infectivity measure and determines the wavelengths of light responsible for the inactivation. The findings presented here provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in aquatic environments and identify solar radiation as a critical process affecting the oocyst survival in the environment.

PCR slippage across the ML-2 microsatellite of the cryptosporidium MIC1 locus enables development of a PCR assay capable of distinguishing the zoonotic cryptosporidium parvum from other human infectious cryptosporidium species

Publisher: Wiley

Date: 17-08-2014

DOI: 10.1111/ZPH.12074

Abstract: Cryptosporidium are ubiquitous and significant enteropathogens of all classes of vertebrates and a major cause of human morbidity and mortality worldwide. Of the 24 recognized species, the zoonotic Cryptosporidium parvum and the host-specific Cryptosporidium hominis cause the majority of cases of human cryptosporidiosis. Here, we report on structural and transcriptional variability between C. parvum and C. hominis at the MIC1 locus, which encodes a microneme localized thrombospondin-like domain containing protein previously demonstrated to be critical for host cell infection by C. parvum. We demonstrate, using reverse transcription quantitative PCR with the aid of genomic data from the EuPathDB site, that the transcribed product in C. hominis is both truncated and significantly down-regulated in the sporozoite. We hypothesize that CpMIC1 may be a genetic factor involved in facilitating the wider host range of C. parvum in comparison with the specific host range of C. hominis. Furthermore, we show that the presence of a microsatellite (ML-2) within the C. parvum MIC-1 locus enables the development of a PCR marker that can rapidly distinguish the zoonotic C. parvum from C. hominis and other significant human infectious Cryptosporidium species due to reproducible PCR slippage across the ML-2 microsatellite. Additionally, we demonstrate that this locus is tightly linked to the GP60 locus, a locus commonly used in the genetic characterization of C. parvum and C. hominis isolates. This marker should provide a robust and additional tool to aid in the rapid identification of C. parvum from other Cryptosporidium species.

Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin

Publisher: Elsevier BV

Date: 05-2003

DOI: 10.1016/S1567-1348(02)00149-1

Abstract: A genetic analysis of Giardia intestinalis, a parasitic protozoan species that is ubiquitous in mammals worldwide, was undertaken using organisms derived from a variety of mammalian hosts in different geographical locations. The test panel of 53 Giardia isolates comprised 48 s les of G. intestinalis, including representatives of all known genetic subgroups, plus an isolate of G. ardeae and four isolates of G. muris. The isolates were compared by allozymic analysis of electrophoretic data obtained for 21 cytosolic enzymes, representing 23 gene loci. Neighbour Joining analysis of the allelic profiles supported the monophyly of G. intestinalis but showed that the species encompasses a rich population substructure. Seven major clusters were evident within G. intestinalis, corresponding to lineages designated previously as genetic assemblages A-G. Some genotypes, e.g. those defining assemblage A, are found in ergent host species and may be zoonotic. However other genotypes, e.g. those defining assemblages C-G, appear to be confined to particular hosts or host groups. The findings reinforce other evidence that G. intestinalis, which was defined on the basis of morphological criteria only, is a species complex.

Risk-based management of drinking water safety in Australia: Implementation of health based targets to determine water treatment requirements and identification of pathogen surrogates for validation o

Publisher: Elsevier BV

Date: 09-2017

DOI: 10.1016/J.FAWPAR.2017.08.002

Development of a nested-PCR assay for the detection of cryptosporidium parvum in finished water

Publisher: Elsevier BV

Date: 05-2001

DOI: 10.1016/S0043-1354(00)00426-7

Abstract: A nested-PCR assay, incorporating an internal positive control, was developed for Cryptosporidium monitoring in finished water. This assay was capable of reproducibly detecting 8 oocysts in spiked-filtered water s les collected from 5 South Australian water treatment plants. The RT-PCR assay of Kaucner and Stinear (Appl. Environ. Microbiol. 64(5) (1998) 1743) was also evaluated for the detection of Cryptosporidium parvum. Initially, under our experimental conditions, a detection level of 27 oocysts was achieved for spiked reagent water s les. This level was improved to 5 oocysts by modification of the method. Untreated South Australian source waters concentrated by calcium carbonate flocculation were found to be highly inhibitory to the RT-PCR assay. Concentration of similar s les using Envirochek filters appeared to eliminate PCR inhibition. While both methods possessed similar sensitivities the nested-PCR assay was more reproducible, more cost effective, simpler to perform and could detect both viable and non-viable intact Cryptosporidium parvum oocysts, which is an important consideration for plant operators. These factors make the nested-PCR assay the method of choice for screening large numbers of potable water s les, where a reliable low level of detection is essential.

Comparison of various staining methods for the detection of Cryptosporidium in cell-free culture

Publisher: Elsevier BV

Date: 09-2008

DOI: 10.1016/J.EXPPARA.2008.04.023

Abstract: The complete development of Cryptosporidium in host cell-free medium first described in 2004, represented a significant advance that can facilitate many aspects of Cryptosporidium research. A current limitation of host cell-free cultivation is the difficulty involved in visualising the life-cycle stages as they are very small in size, morphologically difficult to identify and dispersed throughout the media. This is in contrast to conventional cell culture methods for Cryptosporidium, where it is possible to focus on the host cells and view the foci of infection on the host cells. In the present study, we compared three specific and three non-specific techniques for visualising Cryptosporidium parvum life-cycle stages in cell-free culture antibody staining using anti-sporozoite and anti-oocyst wall antibodies (Sporo-Glo and Crypto Cel), fluorescent in-situ hybridization (FISH) using a Cryptosporidium specific rRNA oligonucleotide probe and the non-specific dyes Texas Red, carboxyfluorescein diacetate succinimidyl ester (CFSE) and 4,6' diamino-2-phenylindole dihydrochloride (DAPI). Results revealed that a combination of Sporo-Glo and Crypto Cel staining resulted in easy and reliable identification of all life-cycle stages.

Detection and significance of the potentially pathogenic amoeboflagellate Naegleria italica in Australia

Publisher: Elsevier BV

Date: 03-2004

DOI: 10.1016/J.PARINT.2003.10.002

Investigating source water Cryptosporidium concentration, species and infectivity rates during rainfall-runoff in a multi-use catchment

Publisher: Elsevier BV

Date: 12-2014

DOI: 10.1016/J.WATRES.2014.08.055

Abstract: Protozoan pathogens present a significant human health concern, and prevention of contamination into potable networks remains a key focus for drinking water providers. Here, we monitored the change in Cryptosporidium concentration in source water during high flow events in a multi-use catchment. Furthermore, we investigated the ersity of Cryptosporidium species/genotypes present in the source water, and delivered an oocyst infectivity fraction. There was a positive and significant correlation between Cryptosporidium concentration and flow (ρ = 0.756) and turbidity (ρ = 0.631) for all rainfall-runoff events, despite variable source water pathogen concentrations. Cell culture assays measured oocyst infectivity and suggested an overall source water infectious fraction of 3.1%. No infectious Cryptosporidium parvum or Cryptosporidium hominis were detected, although molecular testing detected C. parvum in 7% of the s les analysed using PCR-based molecular techniques. Twelve Cryptosporidium species/genotypes were identified using molecular techniques, and were reflective of the host animals typically found in remnant vegetation and agricultural areas. The inclusion of molecular approaches to identify Cryptosporidium species and genotypes highlighted the ersity of pathogens in water, which originated from various sources across the catchment. We suggest this mixing of runoff water from a range of landuses containing erse Cryptosporidium hosts is a key explanation for the often-cited difficulty forming strong pathogen-indicator relationships.

Benthic cyanobacteria: A source of cylindrospermopsin and microcystin in Australian drinking water reservoirs

Publisher: Elsevier BV

Date: 11-2017

DOI: 10.1016/J.WATRES.2017.07.073

Abstract: Cyanobacteria represent a health hazard worldwide due to their production of a range of highly potent toxins in erse aquatic environments. While planktonic species have been the subject of many investigations in terms of risk assessment, little is known about benthic forms and their impact on water quality or human and animal health. This study aimed to purify isolates from environmental benthic biofilms s led from three different drinking water reservoirs and to assess their toxin production by using the following methods: Enzyme-Linked Immunosorbent Assay (ELISA), High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and quantitative PCR (qPCR). Microscopic observation of the isolates allowed the identification of various filamentous cyanobacterial genera: Anabaena (benthic form), Calothrix and Nostoc from the Nostocales and Geitlerinema, Leptolyngbya, Limnothrix, Lyngbya, Oxynema, Phormidium and Pseudanabaena representing non-heterocystous filamentous cyanobacteria. The Phormidium ambiguum strain AWQC-PHO021 was found to produce 739 ng/mg of dry weight (d/w) of cylindrospermopsin and 107 ng/mg (d/w) of deoxy-cylindrospermopsin. The Nostoc linckia strain AWQC-NOS001 produced 400 ng/mg (d/w) of a microcystin analogue. This is the first report of hepatotoxin production by benthic cyanobacteria in temperate Australian drinking water reservoirs. These findings indicate that water quality monitoring programs need to consider benthic cyanobacteria as a potential source of toxins.

Evaluation of Type Replacement Following HPV16/18 Vaccination: Pooled Analysis of Two Randomized Trials

Publisher: Oxford University Press (OUP)

Date: 2017

DOI: 10.1093/JNCI/DJW300

Molecular and phylogenetic analysis of Cryptosporidium muris from various hosts

Publisher: Cambridge University Press (CUP)

Date: 05-2000

DOI: 10.1017/S0031182099005703

Abstract: Isolates of Cryptosporidium muris and C. serpentis were characterized from different hosts using nucleotide sequence analysis of the rDNA 18S and ITS1 regions, and the heat-shock (HSP-70) gene. Phylogenetic analysis confirmed preliminary evidence that C. muris is not a uniform species. Two distinct genotypes were identified within C. muris (1) C. muris genotype A comprising bovine and camel isolates of C. muris from different geographical locations, and (2) C. muris genotype B comprising C. muris isolates from mice, a hamster, a rock hyrax and a camel from the same enclosure. These 2 genotypes may represent separate species but further biological and molecular studies are required for confirmation.

Critical processes affecting Cryptosporidium oocyst survival in the environment

Publisher: Cambridge University Press (CUP)

Date: 13-11-2007

DOI: 10.1017/S0031182006001491

Integrated Cryptosporidium Assay To Determine Oocyst Density, Infectivity, and Genotype for Risk Assessment of Source and Reuse Water

Publisher: American Society for Microbiology

Date: 15-05-2015

DOI: 10.1128/AEM.00163-15

Abstract: Cryptosporidium continues to be problematic for the water industry, with risk assessments often indicating that treatment barriers may fail under extreme conditions. However, risk analyses have historically used oocyst densities and not considered either oocyst infectivity or species/genotype, which can result in an overestimation of risk if the oocysts are not human infective. We describe an integrated assay for determining oocyst density, infectivity, and genotype from a single-s le concentrate, an important advance that overcomes the need for processing multiple-grab s les or splitting s le concentrates for separate analyses. The assay incorporates an oocyst recovery control and is compatible with standard primary concentration techniques. Oocysts were purified from primary concentrates using immunomagnetic separation prior to processing by an infectivity assay. Plate-based cell culture was used to detect infectious foci, with a monolayer washing protocol developed to allow recovery and enumeration of oocysts. A simple DNA extraction protocol was developed to allow typing of any wells containing infectious Cryptosporidium . Water s les from a variety of source water and wastewater matrices, including a semirural catchment, wastewater, an aquifer recharge site, and storm water, were analyzed using the assay. Results demonstrate that the assay can reliably determine oocyst densities, infectivity, and genotype from single-grab s les for a variety of water matrices and emphasize the varying nature of Cryptosporidium risk extant throughout source waters and wastewaters. This assay should therefore enable a more comprehensive understanding of Cryptosporidium risk for different water sources, assisting in the selection of appropriate risk mitigation measures.

Cryptosporidium attenuation across the wastewater treatment train: Recycled water fit for purpose

Publisher: American Society for Microbiology

Date: 03-2017

DOI: 10.1128/AEM.03068-16

Abstract: Compliance with guideline removal targets for Cryptosporidium which do not provide any credit for the inactivation of oocysts through wastewater treatment processes can considerably increase the cost of providing recycled water. Here we present the application of an integrated assay to quantify both oocyst numbers and infectivity levels after various treatment stages at three Victorian and two South Australian (SA) wastewater treatment plants (WWTPs). Oocyst density in the raw sewage was commensurate with community disease burden, with early rounds of s ling capturing a widespread cryptosporidiosis outbreak in Victoria. The level of infectivity of oocysts in sewage was stable throughout the year but was significantly lower at the SA WWTPs. Removals across secondary treatment processes were seasonal, with poorer removals associated with inflow variability however, no decrease in the oocyst infectivity was identified. For SA WWTPs, those oocysts remaining within the secondary treatment-clarified effluent were proportionally more infectious than those in raw sewage. Lagoon systems demonstrated significant inactivation or removal of oocysts, with attenuation being seasonal. Examination of a UV system emphasized its efficacy as a disinfectant barrier but conversely confirmed the importance of a multibarrier approach with the detection of infectious oocysts postdisinfection. The ability to characterize risk from infectious oocysts revealed that the risk from Cryptosporidium is significantly lower than previously thought and that its inclusion in quantitative risk assessments of reuse systems will more accurately direct the selection of treatment strategies and capital expenditure, influencing the sustainability of such schemes. IMPORTANCE Here we present the application of a recently developed integrated assay not only to quantify the removal of Cryptosporidium oocysts but also to quantify their infectivity across various treatment stages at five wastewater treatment plants (WWTPs), thereby better measuring the “true effect” of the treatment train on oocyst risk reduction. For a number of the WWTPs analyzed in this study the risk, is significantly lower than previously thought. Therefore, the inclusion of oocyst infectivity in guideline values and in quantitative microbial risk assessment (QMRA) has the potential to affect future treatment directions and capital expenditure.

Rapid, sensitive, and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis

Publisher: American Society for Microbiology

Date: 09-2006

DOI: 10.1128/AEM.00113-06

Abstract: The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species ( Naegleria fowleri , Naegleria italica , and Naegleria australiensis ) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna . Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.

Solar Radiation Induces Non-Nuclear Perturbations and a False Start to Regulated Exocytosis in Cryptosporidium parvum

Publisher: Public Library of Science (PLoS)

Date: 23-07-2010

DOI: 10.1371/JOURNAL.PONE.0011773

Complete development and multiplication of Cryptosporidium hominis in cell-free culture

Publisher: Elsevier BV

Date: 04-2010

DOI: 10.1016/J.VETPAR.2009.12.021

Abstract: The present study reports for the first time the completion of the life cycle of Cryptosporidium hominis in cell-free culture and multiplication of the parasite via qPCR. In idual life-cycle stages were characterised using Cryptosporidium-specific antibody staining (Sporo-Glo) and fluorescent in situ hybridisation (FISH) staining on cultures inoculated with excysted oocysts and purified sporozoites. In both cultures, C. hominis successfully proliferated and completed its life cycle, however development in cultures inoculated with purified sporozoites lagged behind cultures inoculated with excysted oocysts. Some novel findings of the study include the visualisation of pairing and multiple associations between various developmental stages in a process similar to syzygy and the formation of Cryptosporidium stages (trophozoites and meronts) inside the oocysts without excystation. qPCR analysis revealed a 5-6-fold lification of parasite DNA. Future studies are required to improve the lification of the parasite. The present study confirms the suitability of this culturing model to support the growth and proliferation of C. hominis (which unlike C. parvum, cannot be readily cultured in small animal models) and will greatly assist in our understanding of the developmental biology of Cryptosporidium, its position within the Apicomplexa and its relationship to gregarine protozoa.

Removal and Inactivation of Cryptosporidium from Water

Publisher: Springer Vienna

Date: 14-09-2013

DOI: 10.1007/978-3-7091-1562-6_13

Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection

Publisher: American Society for Microbiology

Date: 05-2003

DOI: 10.1128/AEM.69.5.2505-2511.2003

Abstract: Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved ( log 10 units) with LP-UV (20 mJ/cm 2 ) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with .1 log 10 unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium . The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.

Toolbox for the sampling and monitoring of benthic cyanobacteria

Publisher: Elsevier BV

Date: 02-2020

DOI: 10.1016/J.WATRES.2019.115222

Abstract: Benthic cyanobacteria are a nuisance because they produce highly potent toxins and taste and odour compounds. Despite this, benthic cyanobacteria remain far less studied than their planktonic counterparts. For ex le, little is known about their growth or the seasonality of their secondary metabolite production. Moreover, s ling and monitoring techniques commonly used for the survey of planktonic species are not necessarily applicable to benthic forms. This study aimed to develop and validate a new s ling device for the routine monitoring of benthic mats. Molecular monitoring techniques were established and validated on environmental s les collected in a South Australian reservoir (SA-L2). A total of eight qPCR assays were applied to s les in order to track seasonal variations in cyanobacteria concentrations and associated secondary metabolite production. Next Generation Sequencing was utilised to conduct a microbial community composition analysis and to select the most appropriate substrate material for the s ling of benthic cyanobacteria. The concentration of the secondary metabolites geosmin and 2-methyl-isoborneol were quantified using High-Performance Liquid Chromatography, and concentrations of key nutrients (N, P) were quantified in water s les. The s ling device designed proved efficient and easy to use in the field. The qPCR assay designed for the lification of the cyanobacterial MIB synthase had a high efficiency with a minimum limit of quantification of 4 cell-equivalents per reaction and identified a potential source of MIB in SA-L2 Reservoir. The peak season for benthic growth and secondary metabolite production was observed in spring. Proportionally, 35% of the variability in water geosmin concentrations can be explained by benthic actinobacterial and cyanobacterial activity, showing that freshwater benthic mats represent a significant source of taste and odour compounds.

Molecular and phylogenetic characterisation of Cryptosporidium from birds

Publisher: Elsevier BV

Date: 03-2001

DOI: 10.1016/S0020-7519(00)00164-8

Abstract: Avian isolates of Cryptosporidium species from different geographic locations were sequenced at two loci, the 18S rRNA gene and the heat shock gene (HSP-70). Phylogenetic analysis of the sequence data provided support for the existence of a new avian species of Cryptosporidium infecting finches and a second species infecting a black duck. The identity of Cryptosporidium baileyi and Cryptosporidium meleagridis as valid species was confirmed. Also, C. baileyi was identified in a number of isolates from the brown quail extending the host range of this species.

Emerging technologies for the detection and genetic characterization of protozoan parasites

Publisher: Elsevier BV

Date: 07-2005

DOI: 10.1016/J.PT.2005.05.012

Abstract: The development and adaptation of new technologies for the genetic characterization and identification of parasites continue to accelerate, providing an increasing number of research and analytical tools. We review emerging technologies that have applications in this area, including real-time PCR and microarrays, and discuss the fundamental principles of some of these technologies and how they are applied to characterize parasites. We give special consideration to the application of genetic data to biological questions, where selection of the most appropriate technique depends on the biological question posed by the investigator.

Metabolic flux analysis of hydrogen production network by Clostridium butyricum W5: Effect of pH and glucose concentrations

Publisher: Elsevier BV

Date: 07-2010

DOI: 10.1016/J.IJHYDENE.2010.04.097

DNA extraction from benthic Cyanobacteria: comparative assessment and optimization

Publisher: Oxford University Press (OUP)

Date: 06-12-2017

DOI: 10.1111/JAM.13332

Abstract: Benthic Cyanobacteria produce toxic and odorous compounds similar to their planktonic counterparts, challenging the quality of drinking water supplies. The biofilm that benthic algae and other micro-organisms produce is a complex and protective matrix. Monitoring to determine the abundance and identification of Cyanobacteria, therefore, relies on molecular techniques, with the choice of DNA isolation technique critical. This study investigated which DNA extraction method is optimal for DNA recovery in order to guarantee the best DNA yield for PCR-based analysis of benthic Cyanobacteria. The conventional phenol-chloroform extraction method was compared with five commercial kits, with the addition of chemical and physical cell-lysis steps also trialled. The efficacy of the various methods was evaluated by measuring the quantity and quality of DNA by UV spectrophotometry and by quantitative PCR (qPCR) using Cyanobacteria-specific primers. The yield and quality of DNA retrieved with the commercial kits was significantly higher than that of DNA obtained with the phenol-chloroform protocol. Kits including a physical cell-lysis step, such as the MO BIO Power Soil and Biofilm kits, were the most efficient for DNA isolation from benthic Cyanobacteria. These commercial kits allow greater recovery and the elimination of dangerous chemicals for DNA extraction, making them the method of choice for the isolation of DNA from benthic mats. They also facilitate the extraction of DNA from benthic Cyanobacteria, which can help to improve the characterization of Cyanobacteria in environmental studies using qPCRs or population composition analysis using next-generation sequencing.

Variation in Giardia: towards a taxonomic revision of the genus

Publisher: Elsevier BV

Date: 02-2009

DOI: 10.1016/J.PT.2008.11.006

Abstract: Taxonomic uncertainty has had a negative impact on our understanding of the epidemiology of Giardia infections, particularly the role of wild and domestic animals as sources of human infection. The lack of morphological criteria for species identification and the failure of cross-infection experiments to unequivocally determine host specificity have largely contributed to this uncertainty. However, over the past ten years, it has been possible not only to demonstrate extensive genetic heterogeneity among Giardia isolates from mammals but also to confirm levels of host specificity that were recognized by early taxonomists when they proposed a series of host-related species that we consider should now be re-established.

Cyst morphology and sequence analysis of the small subunit rDNA and ef1α identifies a novel Giardia genotype in a quenda (Isoodon obesulus) from Western Australia

Publisher: Elsevier BV

Date: 12-2004

DOI: 10.1016/J.MEEGID.2004.05.003

Biosynthesis of 2-Methylisoborneol in Cyanobacteria

Publisher: American Chemical Society (ACS)

Date: 21-12-2010

DOI: 10.1021/ES102992P

Development and Evaluation of Three Real-Time PCR Assays for Genotyping and Source Tracking Cryptosporidium spp. in Water

Publisher: American Society for Microbiology

Date: 09-2015

DOI: 10.1128/AEM.01699-15

Abstract: The occurrence of Cryptosporidium oocysts in drinking source water can present a serious public health risk. To rapidly and effectively assess the source and human-infective potential of Cryptosporidium oocysts in water, sensitive detection and correct identification of oocysts to the species level (genotyping) are essential. In this study, we developed three real-time PCR genotyping assays, two targeting the small-subunit (SSU) rRNA gene (18S-LC1 and 18S-LC2 assays) and one targeting the 90-kDa heat shock protein (hsp90) gene (hsp90 assay), and evaluated the sensitivity and Cryptosporidium species detection range of these assays. Using fluorescence resonance energy transfer probes and melt curve analysis, the 18S-LC1 and hsp90 assays could differentiate common human-pathogenic species ( C. parvum , C. hominis , and C. meleagridis ), while the 18S-LC2 assay was able to differentiate nonpathogenic species (such as C. andersoni ) from human-pathogenic ones commonly found in source water. In sensitivity evaluations, the 18S-LC2 and hsp90 genotyping assays could detect as few as 1 Cryptosporidium oocyst per s le. Thus, the 18S-LC2 and hsp90 genotyping assays might be used in environmental monitoring, whereas the 18S-LC1 genotyping assay could be useful for genotyping Cryptosporidium spp. in clinical specimens or wastewater s les.

Effect of water treatment processes on Cryptosporidium infectivity

Publisher: Elsevier BV

Date: 03-2008

DOI: 10.1016/J.WATRES.2007.11.008

Abstract: Conventional water treatment processes have the ability to remove Cryptosporidium oocysts through coagulation, flocculation, sedimentation and filtration, provided there is efficient management of plant performance. The potential exists for the breakthrough of oocysts through the treatment train. The effect of the water treatment chemical aluminium sulphate (alum) on Cryptosporidium oocyst infectivity has been assessed using an assay that combines cell culture and real-time polymerase chain reaction techniques. The infectivity of fresh and temperature-aged oocysts (stored up to 6 months at 4 or 15 degrees C) was unaffected by exposure to a range of doses of alum in standard jar test procedures and dissolved air flotation processes and subsequent exposure to chlorine or chloramine. Removal efficiencies and infectivity measures are important in determining risk to public health and will reflect the ability of water treatment plants to act as a barrier to these pathogens.

Isolates of ‘Candidatus Nostocoida limicola’ Blackall et al. 2000 should be described as three novel species of the genus Tetrasphaera, as Tetrasphaera jenkinsii sp. nov., Tetrasphaera vanveenii sp. n

Publisher: Microbiology Society

Date: 10-2006

DOI: 10.1099/IJS.0.63978-0

Abstract: Despite differences in their morphologies, comparative analyses of 16S rRNA gene sequences revealed high levels of similarity ( %) between strains of the filamentous bacterium ‘ Candidatus Nostocoida limicola’ and the cocci Tetrasphaera australiensis and Tetrasphaera japonica and the rod Tetrasphaera elongata , all isolated from activated sludge. These sequence data and their chemotaxonomic characters, including cell wall, menaquinone and lipid compositions and fingerprints of their 16S–23S rRNA intergenic regions, support the proposition that these isolates should be combined into a single genus containing six species, in the family Intrasporangiaceae in the Actinobacteria . This suggestion receives additional support from DNA–DNA hybridization data and when partial sequences of the rpoC1 gene are compared between these strains. Even though few phenotypic characterization data were obtained for these slowly growing isolates, it is proposed, on the basis of the extensive chemotaxonomic and molecular evidence presented here, that ‘ Candidatus N. limicola’ strains Ben 17, Ben 18, Ben 67, Ben 68 and Ben 74 all be placed into the species Tetrasphaera jenkinsii sp. nov. (type strain Ben 74 T =DSM 17519 T =NCIMB 14128 T ), ‘ Candidatus N. limicola’ strain Ben 70 into Tetrasphaera vanveenii sp. nov. (type strain Ben 70 T =DSM 17518 T =NCIMB 14127 T ) and ‘ Candidatus N. limicola’ strains Ver 1 and Ver 2 into Tetrasphaera veronensis sp. nov. (type strain Ver 1 T =DSM 17520 T =NCIMB 14129 T ).

Evaluation of chromogenic technologies for use in Australian potable water

Publisher: Oxford University Press (OUP)

Date: 10-2008

DOI: 10.1111/J.1365-2672.2008.03842.X

Abstract: To compare the use of MI agar, Membrane Lactose Glucoronide Agar (MLGA), CM1046 agar and Colilert-18 (Defined Substrate Technology, IDEXX Laboratories Pty. Ltd., Sydney) on Australian potable water. Both potable (n = 369) and nonpotable waters (n = 35) were analysed by membrane filtration using chromogenic agars as well as Colilert-18 over a period of 12 months. Recoveries of stressed organisms on these chromogenic media were also investigated. Agar-based chromogenic technologies compared favourably to Colilert-18 for chlorinated waters, but there are possible limitations when using these agars for chloraminated waters. Additionally, the breakthrough of problematic organisms, especially oxidase positive organisms, may lead to misrepresentation or over-estimation of E. coli and total coliforms, particularly on MLGA and CM1046. The recovery of stressed organisms was favoured in the Colilert-18 system when compared to chromogenic agars. MI agar performed better than the other chromogenic agars with respect to recovery and colour identification and discrimination of organisms, and compared favourably with Colilert-18. The use of chromogenic agars in chloraminated waters should be done cautiously. This study provides comparison data for laboratories looking to adopt chromogenic technologies, and is especially important for Australian laboratories wanting to uptake the use of MI agar (as used in USEPA method 1604) for routine use and for gaining accreditation. Additionally, to the best of our knowledge, this is the first reported evaluation of these agars in chloraminated waters and is especially timely as the use of this disinfection agent is increasing.

Legionella confirmation using real-time PCR and SYTO9 is an alternative to current methodology

Publisher: American Society for Microbiology

Date: 12-2005

DOI: 10.1128/AEM.71.12.8944-8948.2005

Abstract: The currently accepted culture techniques for the detection of Legionella spp. in water s les (AS/NZS 3896:1998 and ISO 11731 standard methods) are slow and laborious, requiring from 7 to 14 days for a result. We describe a fully validated rapid confirmation technique that uses real-time PCR incorporating the intercalating dye SYTO9 for the direct identification of primary cultures, significantly decreasing turnaround time and allowing faster remedial action to be taken by the industry.

Identification of polymorphic genes for use in assemblage B genotyping assays through comparative genomics of multiple assemblage B Giardia duodenalis isolates

Publisher: Elsevier BV

Date: 05-2015

DOI: 10.1016/J.MOLBIOPARA.2015.05.002

Abstract: Giardia duodenalis assemblage B is potentially a zoonotic parasite. The characterisation and investigation of isolates has been h ered by greater genetic ersity of assemblage B, limiting the application and utility of current genotyping loci. Since whole genome sequencing is the optimal high-throughput method for gene identification, the present study sequenced assemblage B isolate BAH15c1 and compared the sequence to the draft GS references to identify polymorphic genes for potential use in genotyping assays. The majority of the genome sequence was conserved between the two isolates, producing 508 contigs of 10.4 Mb with 4968 genes. Seventy polymorphic genes for potential use in genotyping assays were identified ranging in variation from elongation factor 1 α, which was the most conserved, through to triose phosphate isomerase, which was the most variable.

Stormwater monitoring using on-line UV-Vis spectroscopy

Publisher: Springer Science and Business Media LLC

Date: 31-10-2021

DOI: 10.1007/S11356-021-17056-7

Genetic manipulation of butyrate formation pathways in Clostridium butyricum

Publisher: Elsevier BV

Date: 09-2011

DOI: 10.1016/J.JBIOTEC.2011.07.004

Abstract: Clostridium butyricum is one of the commonly used species for fermentative hydrogen production. While producing H₂, it can produce acids (lactic, acetic and butyric acids) and CO₂, as well as a small amount of ethanol. It has been proposed that elimination of competing pathways, such as the butyrate formation pathway, should increase H₂ yields in Clostridium species. However, the application of this strategy has been hindered by the unavailability of genetic tools for these organisms. In this study, we successfully transferred a plasmid (pMTL007) to C. butyricum by inter-specific conjugation with Escherichia coli and disrupted hbd, the gene encoding β-hydroxybutyryl-CoA dehydrogenase in C. butyricum. Fermentation data showed that inactivation of hbd in C. butyricum eliminated the butyrate formation pathway, resulting in a significant increase in ethanol production and an obvious decrease in H₂ yield compared with the wild type strain. However, under low partial pressure of H₂, the hbd-deficient strain showed increased H₂ production with the simultaneous decrease of ethanol production, indicating that H₂ production by C. butyricum may compete for NADH with the ethanol formation pathway. Together with the discovery of a potential bifurcating hydrogenase, this study extends our understanding of the mechanism of H₂ production by C. butyricum.

Biochemical kinetics of fermentative hydrogen production by Clostridium butyricum W5

Publisher: Elsevier BV

Date: 2009

DOI: 10.1016/J.IJHYDENE.2008.11.023

A comparative study of carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl ester as indicators of bacterial activity

Publisher: Elsevier BV

Date: 03-2003

DOI: 10.1016/S0167-7012(02)00207-5

Abstract: Staining bacteria with esterified fluorogenic substrates followed by flow cytometric analysis offers a means for rapid detection of metabolically active bacteria. Flow cytometry (FCM) was used to assess carboxyfluorescein diacetate (CFDA) and carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) as indicators of bacterial activity for cultured bacteria, including Aeromonas hydrophila, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis and bacteria from environmental waters. In theory, CFDA/SE should be a better indicator of metabolic bacterial activity compared to CFDA due to greater intracellular retention of the fluorescent product. Qualitative and quantitative analysis of exponential phase cultures, mixtures of active and inactive cells and bacteria from environmental waters revealed CFDA was successful in detecting active bacteria, whereas CFDA/SE was not. CFDA/SE labelled inactive cells with intensities equal to that of the active population and could not even discriminate between bacteria in exponential phase growth and a fixed cell preparation. We propose that the specific mode of action of the succinimidyl ester (SE) group in combination with the nonenzymatic aqueous hydrolysis of the CFDA moiety results in the nonspecific labelling of all cells, irrespective of their metabolic state. This study shows that CFDA/SE is a poor marker of bacterial activity.

Complete development of Cryptosporidium parvum in host cell-free culture

Publisher: Elsevier BV

Date: 06-2004

DOI: 10.1016/J.IJPARA.2004.04.001

Organoids and bioengineered intestinal models: Potential solutions to the Cryptosporidium culturing dilemma

Publisher: MDPI AG

Date: 11-05-2020

DOI: 10.3390/MICROORGANISMS8050715

Abstract: Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes.

Novel primer sets for next generation sequencing-based analyses of water quality

Publisher: Public Library of Science (PLoS)

Date: 24-01-2017

DOI: 10.1371/JOURNAL.PONE.0170008

Molecular systematics of the parasitic protozoan Giardia intestinalis

Publisher: Oxford University Press (OUP)

Date: 09-1999

DOI: 10.1093/OXFORDJOURNALS.MOLBEV.A026204

Abstract: The long-standing controversy regarding whether Giardia intestinalis is a single species prevalent in both human and animal hosts or a species complex consisting of morphologically similar organisms that differ in host range and other biotypic characteristics is an issue with important medical, veterinary, and environmental management implications. In the past decade, highly distinct genotypes (some apparently confined to particular host groups) have been identified by genetic analysis of s les isolated from different host species. The aim of this study was to undertake a phylogenetic analysis of G. intestinalis that were representative of all known major genetic groups and compare them with other Giardia species, viz. G. ardeae, G. muris, and G. microti. Segments from four "housekeeping" genes (specifying glutamate dehydrogenase, triose phosphate isomerase, elongation factor 1 alpha, and 18S ribosomal RNA) were examined by analysis of 0.48-0.69-kb nucleotide sequences determined from DNA lified in polymerase chain reactions from each locus. In addition, isolates were compared by allozymic analysis of electrophoretic data obtained for 21 enzymes representing 23 gene loci. The results obtained from these independent techniques and different loci were essentially congruous. Analyses using G. ardeae and/or G. muris as outgroups supported the monophyly of G. intestinalis and also showed that this species includes genotypes that represent at least seven deeply rooted lineages, herein designated assemblages A-G. Inclusion of G. microti in the analysis of 18S rRNA sequence data demonstrated the monophyly of Giardia with the same median body morphology but did not support the monophyly of G. intestinalis, instead placing G. microti within G. intestinalis. The findings support the hypothesis that G. intestinalis is a species complex and suggest that G. microti is a member of this complex.

Disposable microfluidic micromixers for effective capture of Cryptosporidium parvum oocysts from water samples

Publisher: Springer Science and Business Media LLC

Date: 27-03-2018

DOI: 10.1186/S13036-018-0095-6

Use of DNA melting simulation software for in silico diagnostic assay design: Targeting regions with complex melting curves and confirmation by real-time PCR using intercalating dyes

Publisher: Springer Science and Business Media LLC

Date: 29-03-2007

DOI: 10.1186/1471-2105-8-107

Abstract: DNA melting curve analysis using double-stranded DNA-specific dyes such as SYTO9 produce complex and reproducible melting profiles, resulting in the detection of multiple melting peaks from a single licon and allowing the discrimination of different species. We compare the melting curves of several Naegleria and Cryptosporidium licons generated in vitro with in silico DNA melting simulations using the programs POLAND and MELTSIM., then test the utility of these programs for assay design using a genetic marker for toxin production in cyanobacteria. The SYTO9 melting curve profiles of three species of Naegleria and two species of Cryptosporidium were similar to POLAND and MELTSIM melting simulations, excepting some differences in the relative peak heights and the absolute melting temperatures of these peaks. MELTSIM and POLAND were used to screen sequences from a putative toxin gene in two different species of cyanobacteria and identify regions exhibiting diagnostic melting profiles. For one of these diagnostic regions the POLAND and MELTSIM melting simulations were observed to be different, with POLAND more accurately predicting the melting curve generated in vitro . Upon further investigation of this region with MELTSIM, inconsistencies between the melting simulation for forward and reverse complement sequences were observed. The assay was used to accurately type twenty seven cyanobacterial DNA extracts in vitro . Whilst neither POLAND nor MELTSIM simulation programs were capable of exactly predicting DNA dissociation in the presence of an intercalating dye, the programs were successfully used as tools to identify regions where melting curve differences could be exploited for diagnostic melting curve assay design. Refinements in the simulation parameters would be required to account for the effect of the intercalating dye and salt concentrations used in real-time PCR. The agreement between the melting curve simulations for different species of Naegleria and Cryptosporidium and the complex melting profiles generated in vitro using SYTO9 verified that the complex melting profile of PCR licons was solely the result of DNA dissociation. Other data outputs from these simulations were also used to identify the melting domains that contributed to the observed melting peaks for each of the different PCR licons.

Thwart fatal infant gut parasite

Publisher: Springer Science and Business Media LLC

Date: 03-2014

DOI: 10.1038/507431C

Is nitrification the only cause of microbiologically induced chloramine decay?

Publisher: Elsevier BV

Date: 2016

DOI: 10.1016/J.WATRES.2015.11.016

Abstract: Ammonia degradation was investigated in three batch reactors with differing initial concentrations of bacteria present in the same filtered water source based on pre-treatment filtration techniques. The potential for the bacterial community to degrade the ammonia present was determined in the absence of monochloramine, simulating a distribution system where a loss of disinfectant residual has occurred. Nitrification was observed in only one of the three batch reactors, whereas rapid microbiologically induced chloramine decay was present in two reactors. Results suggest that the microbial decay factor is not a valid tool for indication of nitrification, but may be used as an indicator of the occurrence of rapid monochloramine decay. Intact bacterial cell numbers did not to correlate with changes in ammonia, nitrite or nitrate concentrations and hence did not correlate with the nitrification observed. Neither use of the microbial decay factor or monitoring of ammonia oxidising prokaryotes provided an early indication for the occurrence of nitrification. Hence, monitoring of ammonia and nitrite would still be the most suitable tool for indicating nitrification.

Validation of activated sludge plant performance for virus and protozoan reduction

Publisher: IWA Publishing

Date: 27-02-2013

DOI: 10.2166/WRD.2013.032

Abstract: Validation studies were undertaken at Adelaide metropolitan wastewater treatment plants to establish the actual log10 reduction values (LRVs) of pathogens (viruses and Cryptosporidium) across activated sludge plants (ASPs) as an alternative to accepting the default values attributed by the Department of Health and Ageing (DHA). Grab s les were collected across a 6-week period and assessed for pathogens (adenovirus and Cryptosporidium) and indicator microorganisms (sulphite-reducing clostridia and F-RNA bacteriophage). Through applying the validation process, the DHA has revised the default value for reduction of viruses with an increase from 0.5 log10 to 1 log10 while the value for protozoa remains at 0.5 log10 based on the combined data for a well-operated and maintained ASP. This provides the basis for considering further work at in idual plants which may allow higher log credits to be obtained on a plant by plant basis.

Biochemistry and genetics of taste- and odor-producing cyanobacteria

Publisher: Elsevier BV

Date: 04-2016

DOI: 10.1016/J.HAL.2015.11.008

Abstract: Cyanobacteria are one of the principal sources of volatile organic compounds (VOCs) which cause offensive taste and odor (T&O) in drinking and recreational water, fish, shellfish and other seafood. Although non-toxic to humans, these T&O compounds severely undermine public trust in these commodities, resulting in substantial costs in treatment, and lost revenue to drinking water, aquaculture, food and beverage and tourist/hospitality industries. Mitigation and control have been hindered by the complexity of the communities and processes which produce and modify T&O events, making it difficult to source-track the major producer(s) and the factors governing VOC production and fate. Over the past decade, however, advances in bioinformatics, enzymology, and applied detection technologies have greatly enhanced our understanding of the pathways, the enzymes and the genetic coding for some of the most problematic VOCs produced by cyanobacteria. This has led to the development of tools for rapid and sensitive detection and monitoring for the VOC production at source, and provided the basis for further diagnostics of endogenous and exogenous controls. This review provides an overview of current knowledge of the major cyanobacterial VOCs, the producers, the biochemistry and the genetics and highlight the current applications and further research needs in this area.

Pathogen and Particle Associations in Wastewater: Significance and Implications for Treatment and Disinfection Processes

Publisher: Elsevier

Date: 2016

DOI: 10.1016/BS.AAMBS.2016.08.001

Molecular epidemiology: A multidisciplinary approach to understanding parasitic zoonoses

Publisher: Elsevier BV

Date: 10-2005

DOI: 10.1016/J.IJPARA.2005.06.008

Abstract: Sound application of molecular epidemiological principles requires working knowledge of both molecular biological and epidemiological methods. Molecular tools have become an increasingly important part of studying the epidemiology of infectious agents. Molecular tools have allowed the aetiological agent within a population to be diagnosed with a greater degree of efficiency and accuracy than conventional diagnostic tools. They have increased the understanding of the pathogenicity, virulence, and host-parasite relationships of the aetiological agent, provided information on the genetic structure and taxonomy of the parasite and allowed the zoonotic potential of previously unidentified agents to be determined. This review describes the concept of epidemiology and proper study design, describes the array of currently available molecular biological tools and provides ex les of studies that have integrated both disciplines to successfully unravel zoonotic relationships that would otherwise be impossible utilising conventional diagnostic tools. The current limitations of applying these tools, including cautions that need to be addressed during their application are also discussed.

Evaluating membrane performance in recycled water treatment plants for assets replacement strategy

Publisher: IWA Publishing

Date: 22-08-2017

DOI: 10.2166/WST.2017.466

Abstract: Membranes are an important barrier used in recycled water treatment plants for pathogen removal. Understanding performance over operational life is important to inform membrane replacement. In this study, full scale virus challenge testing was conducted on newly commissioned membranes to validate virus log removal values for accreditation. After six years of operation, the membrane integrity was repeated to ensure compliance with the state regulatory health authority and gain an understanding of the asset's condition. Membrane performance was assessed using a combination of complementary tests including membrane autopsy and chemical tolerance testing to assess in idual modules and selected membrane fibres, followed by a full scale virus challenge for whole of unit assessment. The results demonstrated that the aged membrane fibres were intact and had not been affected by long-term exposure to chlorine, which provides valuable information for membrane asset replacement strategies.

Cryptosporidium spp. in Domestic Dogs: the “Dog” Genotype

Publisher: American Society for Microbiology

Date: 05-2000

DOI: 10.1128/AEM.66.5.2220-2223.2000

Abstract: Genetic and phylogenetic characterization of Cryptosporidium isolates at two loci (18S rRNA gene and heat shock gene) from both Australian and United States dogs demonstrated that dog-derived Cryptosporidium isolates had a distinct genotype which is conserved across geographic areas. Phylogenetic analysis provided support for the idea that the “dog” genotype is, in fact, a valid species.

A molecular phylogeny of nuclear and mitochondrial sequences in Hymenolepis nana (Cestoda) supports the existence of a cryptic species

Publisher: Cambridge University Press (CUP)

Date: 06-2002

DOI: 10.1017/S0031182002002366

Abstract: Since isolates of Hymenolepis nana infecting humans and rodents are morphologically indistinguishable, the only way they can be reliably identified is by comparing the parasite in each host using molecular tools. In the current study, isolates of H. nana from rodent and human hosts from a broad geographical range were sequenced at the ribosomal first internal transcribed spacer (ITS1), the mitochondrial cytochrome c oxidase subunit 1 (C01) gene and the nuclear paramyosin gene loci. Twenty-three isolates of H. nana were sequenced at the ITS1 locus and this confirmed the existence of spacers which, although similar in length (approximately 646 bp), differed in their primary sequences which led to the separation of the isolates into 2 clusters when analysed phylogenetically. This sequence variation was not, however, related to the host of origin of the isolate, thus was not a marker of genetic distinction between H. nana from rodents and humans. Sequencing of a 444 bp fragment of the mitochondrial cytochrome c oxidase 1 gene (C01) in 9 isolates of H. nana from rodents and 6 from humans identified a phylogenetically supported genetic ergence of approximately 5% between some mouse and human isolates. This suggests that H. nana is a species complex, or 'cryptic' species (=morphologically identical yet genetically distinct). A small segment of the nuclear gene, paramyosin, (625 bp or 840 bp) was sequenced in 4 mouse and 3 human isolates of H. nana. However, this gene did not provide the level of heterogeneity required to distinguish between isolates from rodent and human hosts. From the results obtained from faster evolving genes, and the epidemiological evidence, we believe that the life-cycle of H. nana that exists in the north-west of Western Australia is likely to involve mainly 'human to human' transmission.

The Use of Cell Culture and Real-time PCR to Assess Disinfection of Cryptosporidium Parvum

Publisher: Elsevier

Date: 2003

DOI: 10.1016/B978-044451351-9/50035-5

CRYPTOSPORIDIUM SUIS N. SP. (APICOMPLEXA: CRYPTOSPORIDIIDAE) IN PIGS (SUS SCROFA)

Publisher: American Society of Parasitologists

Date: 08-2004

DOI: 10.1645/GE-202R1

The Public Health Significance of Cryptosporidium in the Environment

Publisher: Elsevier

Date: 2003

DOI: 10.1016/B978-044451351-9/50039-2

Virus removal by ultrafiltration: Understanding long-term performance change by application of Bayesian analysis

Publisher: Elsevier BV

Date: 10-2017

DOI: 10.1016/J.WATRES.2017.05.057

Abstract: Ultrafiltration is an effective barrier to waterborne pathogens including viruses. Challenge testing is commonly used to test the inherent reliability of such systems. Performance validation seeks to demonstrate the adequate reliability of the treatment system. Appropriate and rigorous data analysis is an essential aspect of validation testing. In this study we used Bayesian analysis to assess the performance of a full-scale ultrafiltration system which was validated and revalidated after five years of operation. A hierarchical Bayesian model was used to analyse a number of similar ultrafiltration membrane skids working in parallel during the two validation periods. This approach enhanced our ability to obtain accurate estimations of performance variability, especially when the s le size of some system skids was limited. This methodology enabled the quantitative estimation of uncertainty in the performance parameters and generation of predictive distributions incorporating those uncertainties. The results indicated that there was a decrease in the mean skid performance after five years of operation of approximately 1 log reduction value (LRV). Interestingly, variability in the LRV also reduced, with standard deviations from the revalidation data being decreased by a mean 0.37 LRV compared with the original validation data. The model was also useful in comparing the operating performance of the various parallel skids within the same year. Evidence of differences was obtained in 2015 for one of the membrane skids. A hierarchical Bayesian analysis of validation data provides robust estimations of performance and the incorporation of probabilistic analysis which is increasingly important for comprehensive quantitative risk assessment purposes.

Disaggregation of colonies of Microcystis (Cyanobacteria): Efficiency of two techniques assessed using an image analysis system

Publisher: Springer Science and Business Media LLC

Date: 03-2004

DOI: 10.1023/B:JAPH.0000044774.38561.44

Comparison of the levels of intra-specific genetic variation within Giardia muris and Giardia intestinalis

Publisher: Elsevier BV

Date: 08-1998

DOI: 10.1016/S0020-7519(98)00097-6

Abstract: The extent of intra-specific genetic variation between isolates of Giardia muris was assessed by allozyme electrophoresis. Additionally, the levels of allozymic variation detected within G. muris were compared with those observed between members of the two major assemblages of the morphologically distinct species Giardia intestinalis. Four isolates of G. muris were analysed. Three (Ad-120, -150, -151) were isolated from mice in Australia, while the fourth (R-T) was isolated from a golden hamster in North America. The 11 isolates of G. intestinalis (Ad-1, -12, -2, -62, representing genetic Groups I and II of Assemblage A and BAH-12, BRIS/87/HEPU/694, Ad-19, -22, -28, -45, -52, representing genetic Groups III and IV of Assemblage B) were from humans in Australia. Intra-specific genetic variation was detected between G. muris isolates at four of the 23 enzyme loci examined. Similar levels of variation were found within the genetic groups that comprise Assemblages A and B of G. intestinalis. These levels of intra-specific variation are similar to those observed within other morphologically-distinct species of protozoan parasites. We suggest that the magnitude of the genetic differences detected within G. muris provides an indication of the range of genetic variation within other species of Giardia and that this can be used as a model to delineate morphologically similar but genetically distinct (cryptic) species within this genus.

Evaluation of heterotrophic plate and chromogenic agar colony counting in water quality laboratories

Publisher: Elsevier BV

Date: 2015

DOI: 10.1016/J.MEX.2015.10.003

Multi-locus analysis of Giardia duodenalis intra-Assemblage B substitution patterns in cloned culture isolates suggests sub-Assemblage B analyses will require multi-locus genotyping with conserved and

Publisher: Elsevier BV

Date: 04-2011

DOI: 10.1016/J.IJPARA.2010.11.007

Abstract: Recent research concerning Giardia duodenalis has focused on resolving possible sub-assemblages within Assemblages A and B to better understand host-specific and zoonotic relationships. In the present study nine cloned, cultured, Assemblage B isolates were used to investigate the intra-Assemblage B substitution patterns of conserved (ssrDNA, ef, h2b, h4) and variable (tpi, gdh, bg) genes to assess their suitability for further application to sub-assemblage analyses. The resolution of each gene was found to be proportional to its substitution rate and for the genetically narrow s le set examined, the variable genes best represented the consensus phylogeny while the conserved genes only established fractions. However it was demonstrated that the spectra of conserved and variable genes were required to ensure accuracy of inferred phylogeny and it was therefore concluded that further research into sub-Assemblage B groups would require a mixture of conserved and variable genes for the multi-locus analyses of this genetically broad assemblage.

Using amplicon sequencing to characterize and monitor bacterial diversity in drinking water distribution systems

Publisher: American Society for Microbiology

Date: 15-09-2015

DOI: 10.1128/AEM.01297-15

Abstract: Drinking water assessments use a variety of microbial, physical, and chemical indicators to evaluate water treatment efficiency and product water quality. However, these indicators do not allow the complex biological communities, which can adversely impact the performance of drinking water distribution systems (DWDSs), to be characterized. Entire bacterial communities can be studied quickly and inexpensively using targeted metagenomic licon sequencing. Here, licon sequencing of the 16S rRNA gene region was performed alongside traditional water quality measures to assess the health, quality, and efficiency of two distinct, full-scale DWDSs: (i) a linear DWDS supplied with unfiltered water subjected to basic disinfection before distribution and (ii) a complex, branching DWDS treated by a four-stage water treatment plant (WTP) prior to disinfection and distribution. In both DWDSs bacterial communities differed significantly after disinfection, demonstrating the effectiveness of both treatment regimes. However, bacterial repopulation occurred further along in the DWDSs, and some end-user s les were more similar to the source water than to the postdisinfection water. Three s le locations appeared to be nitrified, displaying elevated nitrate levels and decreased ammonia levels, and nitrifying bacterial species, such as Nitrospira , were detected. Burkholderiales were abundant in s les containing large amounts of monochloramine, indicating resistance to disinfection. Genera known to contain pathogenic and fecal-associated species were also identified in several locations. From this study, we conclude that metagenomic licon sequencing is an informative method to support current compliance-based methods and can be used to reveal bacterial community interactions with the chemical and physical properties of DWDSs.

Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community

Publisher: Cambridge University Press (CUP)

Date: 03-2004

DOI: 10.1017/S0031182003004505

Abstract: Giardia duodenalis isolates recovered from humans and dogs living in the same locality in a remote tea-growing community of northeast India were characterized at 3 different loci the SSU-rDNA, elongation factor 1-alpha (ef1-α) and triose phosphate isomerase (tpi) gene. Phylogenetic analysis of the SSU-rDNA and ef1-α genes provided poor genetic resolution of the isolates within various assemblages, stressing the importance of using multiple loci when inferring genotypes to Giardia . Analysis of the tpi gene provided better genetic resolution and placed canine Giardia isolates within the genetic groupings of human isolates (Assemblages A and B). Further evidence for zoonotic transmission was supported by epidemiological data showing a highly significant association between the prevalence of Giardia in humans and presence of a Giardia -positive dog in the same household (odds ratio 3·01, 95% CI, 1·11, 8·39, P =0·0000).

Culture-independent techniques for rapid detection of bacteria associated with loss of chloramine residual in a drinking water system

Publisher: American Society for Microbiology

Date: 11-2005

DOI: 10.1128/AEM.71.11.6479-6488.2005

Abstract: Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 × 10 5 bacteria ml −1 . The bacterial ersity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira -related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene 3.43 × 10 3 active AOB ml −1 were detected in the membrane-intact fraction, and 1.40 × 10 4 active AOB ml −1 were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml −1 detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event.

Assessing the impact of water treatment on bacterial biofilms in drinking water distribution systems using high-throughput DNA sequencing

Publisher: Elsevier BV

Date: 12-2014

DOI: 10.1016/J.CHEMOSPHERE.2014.06.077

Abstract: Biofilm control in drinking water distribution systems (DWDSs) is crucial, as biofilms are known to reduce flow efficiency, impair taste and quality of drinking water and have been implicated in the transmission of harmful pathogens. Microorganisms within biofilm communities are more resistant to disinfection compared to planktonic microorganisms, making them difficult to manage in DWDSs. This study evaluates the impact of four unique drinking water treatments on biofilm community structure using metagenomic DNA sequencing. Four experimental DWDSs were subjected to the following treatments: (1) conventional coagulation, (2) magnetic ion exchange contact (MIEX) plus conventional coagulation, (3) MIEX plus conventional coagulation plus granular activated carbon, and (4) membrane filtration (MF). Bacterial biofilms located inside the pipes of each system were s led under sterile conditions both (a) immediately after treatment application ('inlet') and (b) at a 1 km distance from the treatment application ('outlet'). Bacterial 16S rRNA gene sequencing revealed that the outlet biofilms were more erse than those s led at the inlet for all treatments. The lowest number of unique operational taxonomic units (OTUs) and lowest ersity was observed in the MF inlet. However, the MF system revealed the greatest increase in ersity and OTU count from inlet to outlet. Further, the biofilm communities at the outlet of each system were more similar to one another than to their respective inlet, suggesting that biofilm communities converge towards a common established equilibrium as distance from treatment application increases. Based on the results, MF treatment is most effective at inhibiting biofilm growth, but a highly efficient post-treatment disinfection regime is also critical in order to prevent the high rates of post-treatment regrowth.

Molecular characterisation of Cryptosporidium and Giardia in cats (Felis catus) in Western Australia

Publisher: Elsevier BV

Date: 08-2015

DOI: 10.1016/J.EXPPARA.2015.05.001

Abstract: Little is known of the prevalence of Cryptosporidium and Giardia in domestic cats in Western Australia and their potential role as zoonotic reservoirs for human infection. In the present study, a total of 345 faecal s les from four different sources were screened for the presence of Cryptosporidium and Giardia by PCR and genotyped by sequence analysis. Oocyst numbers and cyst numbers for Cryptosporidium and Giardia respectively were also determined using quantitative PCR assays. Cryptosporidium and Giardia were detected in 9.9% (95% CI 6.7-13.0) and 10.1% (95% CI 7.0-13.3) of cats in Western Australia respectively. Sequence analysis at the 18S rRNA locus identified five Cryptosporidium species/genotypes C. felis (n = 8), C. muris (n = 1), C. ryanae (n = 1), Cryptosporidium rat genotype III (n = 5) and a novel genotype most closely related to Cryptosporidium rat genotype III in one isolate. This is the first report of C. ryanae and Cryptosporidium rat genotype III in cats. For Giardia, assemblage F the most commonly identified species, while only 1 assemblage sequence was detected. Since most human cases of cryptosporidiosis are caused by C. parvum and C. hominis and human cases of giardiasis are caused by G. duodenalis assemblage A and B, the domestic cats in the present study are likely to be of low zoonotic risk to pet owners in Perth. Risk analyses identified that elderly cats (more than 6 years) were more prone to Cryptosporidium and Giardia infections than kittens (less than 6 months) (P = 0.009). Clinical symptoms were not associated with the prevalence of Cryptosporidium and Giardia infections in cats.

Humans, dogs and parasitic zoonoses - Unravelling the relationships in a remote endemic community in northeast India using molecular tools

Publisher: Springer Science and Business Media LLC

Date: 07-2003

DOI: 10.1007/S00436-003-0925-3

Abstract: Canine parasitic zoonoses pose a continuing public health problem, especially in developing countries and communities that are socioeconomically disadvantaged. Our study combined the use of conventional and molecular epidemiological tools to determine the role of dogs in transmission of gastrointestinal (GI) parasites such as hookworms, Giardiaand Ascarisin a parasite endemic tea-growing community in northeast India. A highly sensitive and specific PCR-RFLP was developed to detect and differentiate the zoonotic species of canine hookworm eggs directly from faeces. This allowed epidemiological screening of canine hookworm species in this community to be conducted with ease and accuracy. Seventy two percent of dogs were found to harbour A. caninum, 60% A. braziliense and 37% harboured mixed infections with both hookworms. No A. ceylanicum was detected in the dog population. The zoonotic potential of canine Giardiawas also investigated by characterising Giardia duodenalisrecovered from humans and dogs living in the same locality and households, at three different loci. Phylogenetic and epidemiological analysis provided compelling evidence to support the zoonotic transmission of canine Giardia. Molecular tools were also used to identify the species of Ascarisegg present in over 30% of dog faecal s les. The results demonstrated the role of dogs as a significant disseminator and environmental contaminator of Ascaris lumbricoidesin communities where promiscuous defecation practices exist. Our study demonstrated the usefulness of combining conventional and molecular parasitological and epidemiological tools to help solve unresolved relationships with regards to parasitic zoonoses.

Evaluation of Oocyst DNA Extraction Methods Using Real-time PCR

Publisher: Elsevier

Date: 2003

DOI: 10.1016/B978-044451351-9/50024-0

Dissection of the hierarchy and synergism of the bile derived signal on Cryptosporidium parvum excystation and infectivity

Publisher: Cambridge University Press (CUP)

Date: 16-08-2012

DOI: 10.1017/S0031182012000984

Expression of the geosmin synthase gene in the cyanobacterium anabaena circinalis AWQC318

Publisher: Wiley

Date: 23-09-2011

DOI: 10.1111/J.1529-8817.2011.01061.X

Abstract: The occurrence of taste and odor episodes attributed to geosmin continues to trouble water utilities worldwide, and only recently have advances been made in our fundamental understanding of the biochemical and genetic mechanisms responsible for the production of geosmin in microorganisms. For the first time, we have examined the expression of the geosmin synthase gene and corresponding geosmin production by Anabaena circinalis Rabenh. ex Bornet et Flahault AWQC318 under conditions of continuous light illumination and the removal of light as a stimulus and demonstrate that the expression of geosmin synthase appears to be constitutive under these conditions. The decrease in geosmin synthase transcription post maximum cell numbers and stationary phase suggests that a decrease in isoprenoid synthesis may occur before a decrease in the transcription of ribosomal units as the process of cell death is initiated.

Metabolic flux network and analysis of fermentative hydrogen production

Publisher: Elsevier BV

Date: 07-2011

DOI: 10.1016/J.BIOTECHADV.2011.02.001

Abstract: Fermentative hydrogen production (FHP) has received a great R & D interest in recent decades, as it offers a potential means of producing H₂ from a variety of renewable resources, even wastewater via a low energy continuous process. Various extracellular metabolites including ethanol, acetate, butyrate and lactate can be produced during the fermentation, building a complex metabolic network of the FHP. Except for the recognition of its complexity, the metabolic flux network has not been well understood. Studies on biochemical reactions and metabolic flux network associated with the FHP in anaerobic fermentation system have only been drawn attention in recent years. This review summarizes the biochemical reactions taking place in the metabolic network of FHP. We discuss how the key operation factors influence metabolism in the FHP process. Recently developed and applied technologies for metabolic flux analysis have been described. Future studies on the metabolic network to enhance fermentative hydrogen production by strict anaerobes are recommended. It is expected that this review can provide useful information in terms of fundamental knowledge and update technology for scientists and research engineers in the field of biological hydrogen production.

Selection of surrogate pathogens and process indicator organisms for pasteurisation of municipal wastewater—A survey of literature data on heat inactivation of pathogens

Publisher: Elsevier BV

Date: 2020

DOI: 10.1016/J.PSEP.2019.11.011

Cryptosporidium - Biotechnological advances in the detection, diagnosis and analysis of genetic variation

Publisher: Elsevier BV

Date: 07-2008

DOI: 10.1016/J.BIOTECHADV.2008.02.003

Abstract: Cryptosporidiosis is predominantly a gastrointestinal disease of humans and other animals, caused by various species of protozoan parasites representing the genus Cryptosporidium. This disease, transmitted mainly via the faecal-oral route (in water or food), is of major socioeconomic importance worldwide. The diagnosis and genetic characterization of the different species and population variants (usually recognised as "genotypes" or "subgenotypes") of Cryptosporidium is central to the prevention, surveillance and control of cryptosporidiosis, particularly given that there is presently no broadly applicable treatment regimen for this disease. Although traditional phenotypic techniques have had major limitations in the specific diagnosis of cryptosporidiosis, there have been major advances in the development of molecular analytical and diagnostic tools. This article provides a concise account of Cryptosporidium and cryptosporidiosis, and focuses mainly on recent advances in nucleic acid-based approaches for the diagnosis of cryptosporidiosis and analysis of genetic variation within and among species of Cryptosporidium. These advances represent a significant step toward an improved understanding of the epidemiology as well as the prevention and control of cryptosporidiosis.

Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP

Publisher: Elsevier BV

Date: 06-2004

DOI: 10.1016/J.MEEGID.2004.02.001

Polyphasic identification of cyanobacterial isolates from Australia

Publisher: Elsevier BV

Date: 08-2014

DOI: 10.1016/J.WATRES.2014.04.023

Abstract: Reliable identification of cyanobacterial isolates has significant socio-economic implications as many bloom-forming species affect the aesthetics and safety of drinking water, through the production of taste and odour compounds or toxic metabolites. The limitations of morphological identification have promoted the application of molecular tools, and encouraged the adoption of combined (polyphasic) approaches that include both microscopy- and DNA-based analyses. In this context, the rapid expansion of available sequence data is expected to allow increasingly reliable identification of cyanobacteria, and ultimately resolve current discrepancies between the two approaches. In the present study morphological and molecular characterisations of cyanobacterial isolates (n = 39), collected from various freshwater sites in Australia, were compared. Sequences were obtained for the small ribosomal subunit RNA gene (16S rDNA) (n = 36), the DNA-dependent RNA polymerase gene (rpoC1) (n = 22), and the phycocyanin operon, with its intergenic spacer region (cpcBA-IGS) (n = 19). Phylogenetic analyses identified three cyanobacterial orders: the Chroococcales (n = 8), Oscillatoriales (n = 6), and Nostocales (n = 25). Interestingly, multiple novel genotypes were identified, with 22% of the strains (17/77) having <95% similarity to available sequences in GenBank. Morphological and molecular data were in agreement at the species level for only 26% of the isolates obtained (10/39), while agreement at the genus level was obtained for 31% (12/39). Confident identification of the remaining 44% of the strains (17/39) beyond the order level was not possible. The present study demonstrates that, despite the taxonomic revisions, and advances in molecular-, and bioinformatics-tools, the lack of reliable morphological features, culture-induced pleomorphism, and proportion of misidentified or poorly described sequences in GenBank, still represent significant factors, impeding the confident identification of cyanobacteria species.

Koorie Heritage Trust Inc

Location: Australia

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University Of South Australia

Location: Australia

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University Of Adelaide

Location: Australia

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South Australian Water Corporation

Location: Australia

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Linkage Projects - Grant ID: LP120200587

Start Date: 11-2012

End Date: 12-2017

Amount: $465,000.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR0354888

Start Date: 01-2004

End Date: 02-2004

Amount: $30,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0347126

Start Date: 09-2003

End Date: 12-2008

Amount: $300,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP100100160

Start Date: 2010

End Date: 06-2014

Amount: $210,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0881472

Start Date: 2008

End Date: 06-2013

Amount: $174,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP130100032

Start Date: 10-2013

End Date: 11-2017

Amount: $310,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP170100096

Start Date: 12-2018

End Date: 12-2023

Amount: $355,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP160101050

Start Date: 11-2016

End Date: 12-2020

Amount: $321,000.00

Funder: Australian Research Council