Grantee Research Project Results
2002 Progress Report: FDP -- Development of Detection and Viability Methods for Waterborne Microsporidia
EPA Grant Number: R828041Title: FDP -- Development of Detection and Viability Methods for Waterborne Microsporidia
Investigators: Sonzogni, William C. , Marshall, Marilyn M. , Hoffman, Rebecca M. , Borchardt, Mark
Current Investigators: Hoffman, Rebecca M. , Sonzogni, William C. , Marshall, Marilyn M. , Borchardt, Mark
Institution: University of Arizona , University of Wisconsin - Madison
Current Institution: University of Wisconsin - Madison , University of Arizona
EPA Project Officer: Hahn, Intaek
Project Period: August 1, 2000 through August 3, 2001
Project Period Covered by this Report: August 1, 2001 through August 3, 2002
Project Amount: $375,037
RFA: Drinking Water (1999) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
This research project focuses on the development of a strategy for the recovery and identification of human pathogenic microsporidia from natural waters. This research project is divided into five major objectives: (1) generation of purified spores for development of analytic methods; (2) optimization/development of an efficient sample collection method; (3) concentration/purification of samples by flow cytometry with cell sorting (FCCS); (4) diagnostic assay/viability testing; and (5) validation of a finished method in natural waters.
Progress Summary:
Recovery of E. intestinalis Spores Using Continuous Flow Centrifugation (CFC). During this project period, the authors tested the ability of the continuous flow centrifuge to concentrate spores spiked into filtered tap water. Standards containing either 100 or 1,000 E. intestinalis spores that had been prelabeled with fluorescein isothiocyanate (FITC)-conjugated polyclonal antibody were prepared using flow cytometry with cell sorting. Spores were spiked into 10 L filtered tap water and concentrated using methods reported in the 2001 Progress Report. Sample concentrates were transferred onto well slides and examined under epifluorescence microscopy. Recovery efficiency using this method averaged 61 percent (sd = 12.2, n = 10) at the 10 spores/L level, and 64 percent (sd = 8.9, n = 10) in samples containing 100 spores/L.
Recovery of E. intestinalis Spores Using CFC Combined With FCCS. Flow cytometric purification of spores from CFC concentrates was evaluated for use with more turbid environmental samples when direct well slide analysis was confounded by increased pellet volume. Standards containing either 10, 100, or 1,000 E. intestinalis spores prelabeled with FITC-conjugated polyclonal antibody were prepared using flow cytometry. Spores were spiked into 10 L filtered tap water and concentrated as described in the 2001 Progress Report. Fluorescent particles in the CFC concentrates were flow-sorted onto well slides and confirmed as E. intestinalis spores using epifluorescence microscopy. Recovery efficiency using this method averaged 44 percent (sd = 11.7, n = 3) at the 1 spores/L level, 63 percent (sd = 9.9, n = 10) at 10 spores/L, and 45 percent (sd = 12.2, n = 10) at 100 spores/L.
Purification of E. intestinalis Spores From Water Using Immunomagnetic Separation (IMS). Eight IMS constructs were evaluated for their ability to purify E. intestinalis spores from water. The most efficient spore capture results were achieved using indirect IMS methods, where water concentrates were labeled with polyclonal antimicrosporidia antibody, and the antibody-labeled spores were purified using IMS particles coated anti-rabbit Ig.
The first approach utilized Dynabeads® coated with goat anti-rabbit Ig. Following incubation with the Dynabead® IMS product, the spores were dissociated from the magnetic particles using the acid dissociation method described in the U.S. Environmental Protection Agency (EPA) Method 1622/23. Purified samples were stained with FITC-conjugated antimicrosporidia antibody and flow-sorted onto well slides. The presence of E. intestinalis spores was confirmed under epifluorescence microscopy. An average of 90.2 percent (sd = 7.3, n = 5) of the 1,000 spores seeded into reagent water were recovered using this method.
A second approach using 200 nm magnetic particles coated with goat anti-rabbit Ig also proved successful in purifying E. intestinalis spores from reagent water and concentrated pond water. Water samples containing either 500 or 1,000 spores labeled with FITC-conjugated antimicrosporidia antibody were incubated with the magnetic particles. Recovered magnetic particles were not subjected to acid dissociation; rather, they were flow-sorted onto well slides using the fluorescent and log side scatter criteria used for flow-sorting E. intestinalis spores alone. Magnetic particles bound to fluorescently labeled spores were sorted onto microscope slides along with the organism, while magnetic particles that were not bound to spores were not sorted. The presence of E. intestinalis spores was confirmed using fluorescence microscopy. Recovery of spores seeded into reagent grade water averaged 79 percent at both the 500 (n = 5) and 1,000 (n = 5) seed level, sd = 3.3 and 6.2, respectively. Average recovery spores seeded into pond water was 67 percent (sd = 4.1, n = 5). The presence of the 200 nm particles on the E. intestinalis spore did not interfere with microscopic visualization of the organism.
Molecular Analysis. Reverse transcription-polymerase chain reaction (RT-PCR) CC assays were conducted using forward and reverse Hsp 70 primer sets (forward 5'-gcgcttgcagatgcgggactgg-3' reverse 5'-gcctttcttctcttcatcacg-3') (Jost BH, University of Arizona). Hsp 70 gene amplification compared favorably with standard cell culture infectivity data and differentiated between live and dead E. cuniculi and E. hellem spores. However, these primers could not differentiate between live and heat-killed E. intestinalis spores (see Table 1).
RT-PCR CC assays using -tubulin primer sets demonstrated an equivalency comparable to the cell culture coverslips at 104 spores for E. intestinalis and E. hellem, but not for E. cuniculi. Table 1 demonstrates the necessity of primer evaluation and assay validation with dead and live spores for cell culture infectivity assays. Thus, to use an RT-PCR assay for water samples or viability testing, it may be necessary to use a nested primer approach, a multiplex approach, or to develop additional primer sets that would identify all three species with one assay. Before this assay is ready for disinfection studies, the assay sensitivity needs to be addressed, as the data in Table 1 are based on 104 spores/well. However, we have demonstrated that the RT-PCR CC assay may become a useful tool for disinfection evaluation assays.
Organism | Live or Dead | 16SRNA | ß-tubulin | Hsp70 |
E. intestinalis | Live | + | + | - |
E. intestinalis | Dead | + | - | - |
E. cuniculi | Live | + | - | + |
E. cuniculi | Dead | + | - | - |
E. hellem | Live | + | + | + |
E. hellem | Dead | + | - | - |
Detection of E. intestinalis Seeded in Natural Water Samples. PCR detection
of E. intestinalis spores seeded into source water using the 16S rRNA
primers was evaluated using three different purification kits: QIAamp®
DNA Mimi Kit [Qiagen Inc., Valencia, CA]; Insta Gene Matrix [Bio-Rad Laboratories,
Life Science Research, Hercules, CA] and Fast DNA®
Kit [Qbiogene, Inc., Carlsbad, CA]. Flow cytometer counted spores [100 spores/10µL]
were seeded into river and pond water at the levels listed in Table 2. Both
the extraction method and water matrix were shown to influence test results.
Extraction Kit | Source Water | No. Spores | No. Positive/Total No. |
QIAamp (Lot #1) | River | 100a | 4/20 |
WSLH Pond 2 | 100b | 3/6 | |
QIAamp (Lot #2) | NanoPure | 100a | 1/6 |
River | 100a | 2/12 | |
InstaGene | WSLH Pond 2 | 100b | 7/8 |
UA Pond 2 | 100a | 3/3 | |
River | 100a | 0/2 | |
FastDNA Kit | NanoPure | 100a | 2/2 |
NanoPure | 50a | 2/3 | |
a Enumerated by hemocytometer. | |||
b Flow counted spores. |
Future Activities:
During the next reporting period, we will determine the efficiency of our IMS approach in purifying E. intestinalis spores spiked into natural water concentrates. We will evaluate the molecular detection of spores concentrated and purified using the continuous flow centrifuge and IMS. Finally, the complete method will be applied to spiked and unspiked natural waters.
Journal Articles:
No journal articles submitted with this report: View all 14 publications for this projectSupplemental Keywords:
microsporidia, Encephalitozoon, protozoa, flow cytometry, continuous flow centrifugation, water., RFA, Scientific Discipline, Water, Environmental Chemistry, Health Risk Assessment, Analytical Chemistry, Environmental Microbiology, Environmental Monitoring, Drinking Water, complex mixtures, natural waters, pathogens, molecular biotechnologies, monitoring, encephalitozoon, microbiological organisms, waterborne disease, exposure and effects, exposure, viability methods, treatment, microbial risk management, oligoprobe, parasites, drinking water contaminants, microsporidia, water treatment, contaminant candidate listProgress 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.