Cryptosporidium Viability And Infectivity Methods

Cell culture techniques are routinely used for measuring the infectivity of a wide range of human pathogens. A variety of different cell culture systems and detection methodologies have been applied to Cryptosporidium parvum. However, the correlation between cell culture methods and animal infectivity assays has not been thoroughly investigated. Although many cell culture methods have been developed for C. parvum, it has not been proven that infectivity in cell culture is a good indicator of the ability of oocysts to cause infections in animals. The objective of this research was to compare in-vitro cell culture methods with a mouse assay for measuring infectivity of C. parvum oocysts. The specific objectives were to (1) compare the dose response and sensitivity of cell culture and mouse assays with multiple isolates; (2) compare infectivity methods with oocysts exposed to environmental water samples; (3) determine the reproducibility and variability of the methods; and (4) compare cell culture and animal assays for assessing ozone and UV disinfection.For untreated oocysts, challenge doses were enumerated by flow cytometry. Dose response curves were constructed by regression analysis of oocyst dose against a logistic transformation of the proportional infectivity and the 50% infectious doses for each isolate were calculated by solving the regression for a logit value of zero. Infections in CD-1 mice were detected by microscopy following staining with hematoxylin and eosin. Infection in HCT-8 and Caco-2 cells was detected by C. parvum-specific RT-PCR. In MDCK cells, infection was detected using immunofluorescence. For disinfection studies, oocysts were exposed to UV using a medium-pressure, collimated beam apparatus and inactivation was measured as the difference in ID50 of unexposed and UV-exposed oocysts. Oocysts were exposed to ozone using batch, semi-batch, and single continuously stirred tank reactors at 1, 5, and 15°C.This investigation demonstrated that in-vitro cell culture was equivalent with a mouse assay for measuring infectivity of untreated C. parvum oocysts and should therefore be considered a practical alternative for assessing the potential of oocysts to cause infection. However, the high levels of variability displayed by mouse and cell culture methods indicated that infectivity and disinfection experiments should be limited to discerning relatively large differences. Of the three cell culture assays, the HCT-8/RT-PCR method displayed the closest agreement with the CD-1 mouse assay. C. hominis was infectious in HCT-8 cells but did not infect mice. Similar results were obtained with CD-1 mice and HCT-8 cells for measuring infectivity of oocysts that had been exposed to environmental water for 35 days. There was also very good agreement between HCT-8 cell culture and CD-1 mouse assays for measuring UV inactivation of C. parvum. A medium-pressure UV dosage of 5.6 mJ/cm2 resulted in 2-log10 inactivation. The shapes of ozone inactivation curves were generally the same for mouse and cell culture derived data although the CD-1 mouse assay typically generated 0.5 to 1-log10 higher levels of inactivation than HCT-8 cells. In addition, there was a stimulatory response in oocysts exposed to ozone below 20 mg.min/L when assayed by HCT-8 cell culture. Consequently, further research is necessary to understand the response of oocysts to ozone when inactivation is assessed by cell culture methods. The water industry should adopt in-vitro cell culture as a routine method for measuring the infectivity of waterborne C. parvum and C. hominis oocysts. This project has demonstrated that cell culture has equivalency with the standard CD-1 mouse assay and cell culture assays can be applied to oocysts recovered from water using approved methods. However, there needs to be a thorough, robust, and well-controlled study to compare the various cell culture-based assays for measuring C. parvum and C. hominis infectivity. This evaluation should include inter-laboratory comparisons and round-robin testing. Cell culture-based assays should also be used to assess disinfection of C. hominis isolates. Originally published by AwwaRF for its subscribers in 2004. This publication can also be purchased and downloaded via Pay Per View on Water Intelligence Online - click on the Pay Per View icon below

From the microscopic observation of infection to the widespread application of molecular techniques in taxonomy and epidemiology, to the genome sequencing of two major species and advances in biochemistry, phylogeny, and water treatment, new information on this fascinating genus continues to mount as we discover and utilize the latest scientific te

This study focused on ideal methods of detecting C. parvum oocysts which give low detection limits, specific and sensitive detection in concentraded samples, and differentiation of viable from non-viable oocysts; are usable by a water treatment facility and provide quantitative results within a few hours. Potential useful methods are continuous flow centrifugation, rel-time TaqMan PCR, cell culture inoculation followed by PCR of DNA extration, and FISH.

Cryptosporidium, in its various forms, is a widely recognised cause of outbreaks of waterborne disease. Regulatory bodies worldwide are increasingly requiring the development of "fit-for-purpose" detection methods for this protozoan parasite, but analysis is often problematic. Bringing together international academic and industry-based experts, this book provides a comprehensive review of the current state of analytical techniques for the detection of Cryptosporidium, as well as looking at likely future developments. In particular, the issues of species identification and oocyst viability are addressed. Quality assurance issues and potential problems associated with the new Cryptosporidium regulations are also highlighted. The extent of the perceived problems and the regulatory backdrop against which the analysis must be carried out are also discussed. Scientists in the water industry, environmental testing laboratories, researchers, consultants, environmental health professionals, food manufacturers and regulatory or environmental bodies are amongst the many who should read this book. In addition, anyone with an interest in microbiological challenges and problem-solving will welcome the coverage.

The second edition of Microbiology of Waterborne Diseases describes the diseases associated with water, their causative agents and the ways in which they gain access to water systems. The book is divided into sections covering bacteria, protozoa, and viruses. Other sections detail methods for detecting and identifying waterborne microorganisms, and the ways in which they are removed from water, including chlorine, ozone, and ultraviolet disinfection. The second edition of this handbook has been updated with information on biofilms and antimicrobial resistance. The impact of global warming and climate change phenomena on waterborne illnesses are also discussed. This book serves as an indispensable reference for public health microbiologists, water utility scientists, research water pollution microbiologists environmental health officers, consultants in communicable disease control and microbial water pollution students. Focuses on the microorganisms of most significance to public health, including E. coli, cryptosporidium, and enterovirus Highlights the basic microbiology, clinical features, survival in the environment, and gives a risk assessment for each pathogen Contains new material on antimicrobial resistance and biofilms Covers drinking water and both marine and freshwater recreational bathing waters

Twort's Water Supply, Seventh Edition, has been expanded to provide the latest tools and techniques to meet engineering challenges over dwindling natural resources. Approximately 1.1 billion people in rural and peri-urban communities of developing countries do not have access to safe drinking water. The mortality from diarrhea-related diseases amounts to 2.2 million people each year from the consumption of unsafe water. This update reflects the latest WHO, European, UK, and US standards, including the European Water Framework Directive. The book also includes an expansion of waste and sludge disposal, including energy and sustainability, and new chapters on intakes, chemical storage, handling, and sampling. Written for both professionals and students, this book is essential reading for anyone working in water engineering. Features expanded coverage of waste and sludge disposal to include energy use and sustainability Includes a new chapter on intakes Includes a new chapter on chemical storage and handling