Development and Evaluation of Methods for the Concentration, Separation, Detection, and Viability/Infectivity of Three Protozoa from Large Volume of Water

EPA Grant Number: R828043
Title: Development and Evaluation of Methods for the Concentration, Separation, Detection, and Viability/Infectivity of Three Protozoa from Large Volume of Water
Investigators: Tzipori, Saul , Buckholt, Michael , Widmer, Giovanni , Zuckermann, Udi
Institution: Tufts University
EPA Project Officer: Nolt-Helms, Cynthia
Project Period: March 1, 2000 through March 1, 2003
Project Amount: $525,000
RFA: Drinking Water (1999) RFA Text |  Recipients Lists
Research Category: Drinking Water , Water


This proposal will develop, evaluate and streamline several analytical methods required to concentrate, separate, identify and test for infectivity and/or viability of important waterborne pathogenic protozoa known to cause disease in humans. They include Cryptosporidium of human (C. parvum, genotype 1) or animals (C. parvum genotype 2 and the avian C. bailey), Giardia lamblia, and the microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis (formerly E. septate).


We will optimize and standardize the portable continuous flow centrifuge device, which we have successfully adapted from a blood cell separator to concentrate C. parvum oocysts, Giardia cysts and microsporidia spores from large volumes of drinking or turbid water. This method will be evaluated under various conditions, which include different matrices and volumes of water and variable numbers of parasites (Objective 1). The bulk of this work will be carried out during year 1.

We will generate monoclonal antibodies (mAb) and rabbit polyclonal antibodies against E. bieneusi, a non-cultivable microsporidia of humans with immunodeficiencies, and against E. intestinals. We have in our laboratory methods to propagate both microsporidia to generate the necessary antigen for immunization of mice (a panel of mAb is currently being produced against E. bieneusi). We will use infected tissue of macaques or piglets to screen E. bieneusi clones, and cell culture-derived spores for E. intestinalis. The antibodies will be labeled with FITC, and as with C. parvum and Giardia, develop paramagnetic beads for immuno- magnetic separation (IMS) detection system for E. bieneusi. E. bieneusi spores (Objective 2).

We will evaluate our newly developed infectivity and molecular (mRNA) viability assays for C. parvum, on oocysts recovered from large volumes of drinking and turbid water, using the flow centrifugation method. For Giardia, we propose to develop a molecular viability assay, similar to C. parvum, and cell culture infectivity assays for the 2 microsporidia (Objective 3).

Expected Results:

The beneficial outcome from this proposal to the EPA will be the availability of a streamlined analytical method that will determine the presence, concentration and infectivity and/or viability of Cryptosporidium, Giardia, E. bieneusi and E. intestinalis in large or small volumes of drinking or raw water. It is likely that this approach can be used to detect other waterborne microorganisms including bacteria, and with the use of antibody-coated magnetic beads, viruses as well.

Publications and Presentations:

Publications have been submitted on this project: View all 4 publications for this project

Journal Articles:

Journal Articles have been submitted on this project: View all 3 journal articles for this project

Supplemental Keywords:

Cryptosporidium, Microsporidia, Enterocytozoon Bieneusi, Encephalitozoon intestinalis, Giardia, protozoa, water, RFA, Health, PHYSICAL ASPECTS, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Health Risk Assessment, Risk Assessments, Monitoring/Modeling, Physical Processes, Drinking Water, public water systems, microbial contamination, enterocytozoon , concentration device, microbial monitoring, monitoring, measurement , detection, waterborne disease, bacteria, microbiological organisms, encephalitozoon, assays, infective dose, exposure and effects, exposure, infectivity assays, cryptosporidium , analytical methods, microbial risk management, measurement, microorganism, pathogenic protozoa, infectivity, Giardia, microsporidia, assessment technology, cryptosporidium

Progress and Final Reports:

  • 2000 Progress Report
  • 2001 Progress Report
  • Final Report