Alexandra Keegan

Environmental Temperature Controls Cryptosporidium Oocyst Metabolic Rate and Associated Retention of Infectivity

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 log10 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.

Cell Culture-Taqman PCR Assay for Evaluation of Cryptosporidium parvum Disinfection

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 (>2 log10 units) with LP-UV (20 mJ/cm2) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log10 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.

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

Aims:  To isolate and identify bacteria from a sand filter column capable of degrading the taste and odour compound, geosmin. In doing so, to investigate if these organisms degrade geosmin either individually or if an alternative mechanism is utilized.

Mycobacterium avium complex - the role of potable water in disease transmission

Mycobacterium avium complex (MAC) is a group of opportunistic pathogens of major public health concern. It is responsible for a wide spectrum of disease dependent on subspecies, route of infection and patients pre‐existing conditions. Presently, there is limited research on the incidence of MAC infection that considers both pulmonary and other clinical manifestations. MAC has been isolated from various terrestrial and aquatic environments including natural waters, engineered water systems and soils. Identifying the specific environmental sources responsible for human infection is essential in minimizing disease prevalence. This paper reviews current literature and case studies regarding the wide spectrum of disease caused by MAC and the role of potable water in disease transmission. Potable water was recognized as a putative pathway for MAC infection. Contaminated potable water sources associated with human infection included warm water distribution systems, showers, faucets, household drinking water, swimming pools and hot tub spas. MAC can maintain long‐term contamination of potable water sources through its high resistance to disinfectants, association with biofilms and intracellular parasitism of free‐living protozoa. Further research is required to investigate the efficiency of water treatment processes against MAC and into construction and maintenance of warm water distribution systems and the role they play in MAC proliferation.

Cryptosporidium cell culture infectivity assay design

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 hampered 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.

Uncertainties associated with assessing the public health risk from Legionella

Legionella is an opportunistic pathogen of public health concern. Current regulatory and management guidelines for the control of this organism are informed by risk assessments. However, there are many unanswered questions and uncertainties regarding Legionella epidemiology, strain infectivity, infectious dose, and detection methods. This review follows the EnHealth Risk Assessment Framework, to examine the current information available regarding Legionella risk and discuss the uncertainties and assumptions. This review can be used as a tool for understanding the uncertainties associated with Legionella risk assessment. It also serves to highlight the areas of Legionella research that require future focus. Improvement of these uncertainties will provide information to enhance risk management practices for Legionella, potentially improving public health protection and reducing the economic costs by streamlining current management practices.

Detection of Legionella, L. pneumophila and Mycobacterium Avium Complex (MAC) along Potable Water Distribution Pipelines

Inhalation of potable water presents a potential route of exposure to opportunistic pathogens and hence warrants significant public health concern. This study used qPCR to detect opportunistic pathogens Legionella spp., L. pneumophila and MAC at multiple points along two potable water distribution pipelines. One used chlorine disinfection and the other chloramine disinfection. Samples were collected four times over the year to provide seasonal variation and the chlorine or chloramine residual was measured during collection. Legionella spp., L. pneumophila and MAC were detected in both distribution systems throughout the year and were all detected at a maximum concentration of 103 copies/mL in the chlorine disinfected system and 106, 103 and 104 copies/mL respectively in the chloramine disinfected system. The concentrations of these opportunistic pathogens were primarily controlled throughout the distribution network through the maintenance of disinfection residuals. At a dead-end and when the disinfection residual was not maintained significant (p < 0.05) increases in concentration were observed when compared to the concentration measured closest to the processing plant in the same pipeline and sampling period. Total coliforms were not present in any water sample collected. This study demonstrates the ability of Legionella spp., L. pneumophila and MAC to survive the potable water disinfection process and highlights the need for greater measures to control these organisms along the distribution pipeline and at point of use.

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

Bile salts have been identified as an important trigger for excystation of Cryptosporidium oocysts but the hierarchy or synergism of this signal in relation to other triggers involved in excystation is poorly understood. In addition to excystation, bile salts have also been reported to increase the invasiveness of sporozoites within in vitro culture, possibly by affecting the secretory pathway via modification of intracellular calcium signalling. Nevertheless, incorporation of bile or bile salts into in vitro assays is not universal, with recent reports of negative effects on parasite growth. Here we report that bile and sodium taurocholate significantly affect both excystation rate and parasite in vitro growth. We demonstrate that their effect on excystation is dose, time and pre-treatment temperature dependent, while increases in parasite replication appear to be associated with modulation of parasite intracellular calcium and increased host cell susceptibility to infection. Notably, we illustrate that bile has a significant effect on host cells and can be cytotoxic at concentrations not much higher than those currently used for in vitro assays. This work should assist with more rational design of in vitro culture systems, with significant considerations for assay format when incorporating bile or bile salts as an excystation trigger.

Pathogen risk indicators for wastewater and biosolids

Direct analysis of potable water for pathogenic microorganisms has generally been avoided by water suppliers because pathogens are frequently present intermittently and in low numbers. Direct analysis for pathogens would require concentration of large sample volumes and more complex analytical procedures both of which are expensive and currently considered not to be more protective of public health than using appropriate pathogen index organisms or surrogates. Human feces contain about 1012 bacteria per gram, hence Escherichia coli is always present in high numbers in domestic wastewater (around 109 cfu/g) and can be detected relatively cheaply by culture methods. Hence, E. coli has become the chosen indicator for fecal pollution of water. While E. coli is a valuable warning indicator in potable water supplies, its value in domestic wastewater and biosolids applications is reduced because the source water and sludge is always fecally polluted. Instead, an indicator more clearly linked to pathogen presence, an index organism, is required. An index organism is defined as a group or species indicative of pathogen presence, such as E. coli as an index organism for Salmonella. An approach is to find indicators which are removed or inactivated similarly to pathogens by wastewater and biosolids treatment processes. Such process indicators, called model organisms or surrogates, are defined as a group of organisms that demonstrate the efficacy of a process). When coupled with data collected over time on the numbers of pathogens in the matrix prior to treatment, can indicate the risk attached to using treated water. As climate change continues to place stress on water resources, communities are increasingly looking to recycled water as a supplementary water source. Hence identification of process indicators for recycled water is becoming imperative so that recycled water can be used appropriately so as to minimize risks. As pathogen reduction in primary and secondary wastewater treatment processes is not as great as in tertiary and disinfection treatment processes, the latter treatment processes have been the focus of this study. Similarly, as disposal of biosolids to landfill sites becomes increasing costly and inorganic fertilizer feedstocks decline, the beneficial use of biosolids is becoming more important. Identification of process indicators for biosolids will encourage these beneficial uses as the risk to human health can be better assessed.

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

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 sample 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.