Grantee Research Project Results
2001 Progress Report: Development and Evaluation of Methods for the Concentration, Separation, Detection, and Viability/Infectivity of Three Protozoa from Large Volume of Water
EPA Grant Number: R828043Title: Development and Evaluation of Methods for the Concentration, Separation, Detection, and Viability/Infectivity of Three Protozoa from Large Volume of Water
Investigators: Tzipori, Saul , Sheoran, Abhineet , Widmer, Giovanni , Zuckermann, Udi
Current Investigators: Tzipori, Saul , Widmer, Giovanni , Buckholt, Michael , Zuckermann, Udi
Institution: Tufts University
EPA Project Officer: Hahn, Intaek
Project Period: March 1, 2000 through March 1, 2003
Project Period Covered by this Report: March 1, 2001 through March 1, 2002
Project Amount: $525,000
RFA: Drinking Water (1999) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
There are three major objectives for this research project:
Objective 1: To evaluate and optimize the modified continuous flow centrifugation (CFC) method for recovery of protozoa (Cryptosporidium spp., Giardia, and Microsporidia spp.) from turbid and large volumes of water. The CFC method allows for concentration of oocysts, cysts, and spores from large volumes of water, and for continuous monitoring of their presence in water, as opposed to a one-time sampling of existing methods. This method is efficient, portable, rapid, and easy to operate.
Objective 2: To develop E. bieneusi and E. intestinalis detection techniques, which include production of specific antibody against both protozoa, with a view to develop specific and sensitive methods using immunomagnetic separation (IMS). Concentration of oocysts, cysts, and spores from raw or large volumes of drinking water samples requires sensitive and specific detection systems. A combined method for oocysts and cysts using IMS already exists. We propose to develop monoclonal antibodies and rabbit polyclonal antibodies against E. bieneusi and E. intestinalis.
Objective 3: To develop, optimize, and evaluate the infectivity and viability assays for Cryptosporidium spp., Giardia and Microsporidia spp., recovered from turbid and large volumes of water. The infectivity and viability of recovered oocysts, cysts, and spores is important in relation to the following questions: (1) is water treatment effective in inactivating these pathogens? (2) do the concentration and separation processes impact infectivity/viability?, and (3) can molecular fingerprinting of oocysts, cysts, and spores help determine the source/origin of contamination?
Progress Summary:
The second phase of this project included optimization of recovery of microsporidia from 10-50 L of water. Recoveries of E. intestinalis, the microsporidia used to optimize the CFC system, ranged from 20 percent (50 L), to more than 90 percent from 10 L. This is greater than any of the current concentration methods for these volumes. The next phase is to try higher volumes and a mixture of pathogens, which will include C. parvum oocysts and Giardia cysts. We have further characterized the monoclonal antibodies against E. intestinalis. They were found to be only highly specific for E. intestinalis. We have been unsuccessful so far in identifying specific monoclonal antibodies against E. bieneusi due to lack of suitable material. The attempts to identify RNA for the development of viability assay for C. parvum, Giardia, and E. bieneusi continue. The most suitable target for C. parvum, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), appears to be the viral dsRNA, which has a fast rate of decay after oocyst inactivation. For Giardia, the cyst wall protein 2 mRNA has been identified as suitable for the RT-PCR reaction. We have attempted to use the small subunit ribosomal RNA of E. bieneusi for the RT-PCR reaction, but again, the lack of suitable and sufficient material has been a problem. As with monoclonal antibody production, we plan to obtain material containing spores from other sources to continue and complete this work over the next 12 months.
Future Activities:
We will try recovery of Microsporidia from higher volumes of water, and a mixture of pathogens including C. parvum oocysts, Giardia cysts, and E. intestinalis spores. We plan to obtain fresh spores from other sources. Some of the material used to produce monoclonal antibodies will be used to complete the viability assay for E. bieneusi. The assays for Giardia and C. parvum also will be completed over the next 12 months. Three publications will result from this work. These include: (1) the optimization of CFC for C. parvum and Giardia, (2) the rate of recovery of microsporidia using the CFC, and (3) the production and characterization of monoclonal antibodies against E. intestinalis.
Journal Articles:
No journal articles submitted with this report: View all 4 publications for this projectSupplemental Keywords:
Cryptosporidium, Microsporidia, E. bieneusi, E. intestianalis, Giardia, protozoa, water., RFA, Health, Ecosystem Protection/Environmental Exposure & Risk, Scientific Discipline, PHYSICAL ASPECTS, Water, Health Risk Assessment, Physical Processes, Risk Assessments, Monitoring/Modeling, Drinking Water, microbial contamination, concentration device, assays, measurement, microbiological organisms, encephalitozoon, microorganism, measurement , microbial risk management, pathogenic protozoa, analytical methods, infectivity assays, monitoring, infectivity, microbial monitoring, assessment technology, exposure, waterborne disease, Giardia, public water systems, cryptosporidium , cryptosporidium, human health risk, infective dose, detection, bacteria, microsporidia, enterocytozoonProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.