Development and Evaluation of Methods for the Concentration, Separation, Detection, and Viability/Infectivity of Three Protozoa from Large Volume of WaterEPA 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
Description: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).
Approach: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).