Grantee Research Project Results
2001 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. , 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, 2000 through August 3, 2001
Project Amount: $375,037
RFA: Drinking Water (1999) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
This study focuses on the development of a strategy for the recovery and identification of human pathogenic microsporidia from natural waters. The research effort is divided into five major objectives: (1) generation of purified spores for development of analytic methods; (2) development/optimization of an efficient sample collection method; (3) sample concentration/purification by flow cytometry; (4) diagnostic assay/viability testing; and (5) validation of a finished method in natural waters.
Progress Summary:
Spores of Encephalitozoon cuniculi, ATCC 50502, E. hellem, ATCC 50451, and a duodenal isolate of E. intestinalis, ATCC 50603, were propagated in rabbit kidney cells (RK-13). Spores were recovered from spent culture medium by centrifugation at 1,000 x g for 10 minutes. Spore pellets were purified from cell debris using a differential density gradient centrifugation (Percoll®). Equal volumes of Percoll® and spore harvest pellet were mixed with sterile water in 15 mL conical polypropylene tubes and the mixtures centrifuged at 2,300 x g for 30 minutes. Spore-containing pellets were washed with sterile NANOpure water (1,000 x g for 10 minutes), the supernatant aspirated, and the pellets resuspended in sterile NANOpure water. After enumeration by hemacytometer, the spore concentration was adjusted to 1 x 107 spores/mL per tube and stored in PBS or sterile NANOpure water at 4ºC.
Flow cytometric analysis of purified spore suspensions produced over a 1-year period (n=9) showed that freshly harvested suspensions were comprised of two subpopulations differing in physical size, permeability to vital dyes, antibody staining with monoclonal antibody 7G7, and infectivity in cell culture assays. These subpopulations were purified using flow cytometry with cell sorting. The small subpopulation, comprising nearly 80 percent of spore total, was noninfectious in cell culture infectivity assays and permeable to vital dyes including SYTOX Green, propidium iodide (PI), and DAPI. The large subpopulation (16 percent of the spore total) was infectious in cell culture infectivity assays and impermeant to all vital dyes with the exception of DAPI when ethanol pretreatment was performed. PCR products of the two flow-sorted subpopulations were sequenced and compared to E. intestinalis sequences for the 16S rRNA or -tubulin genes. The sequence data indicated that both of the subpopulations possessed the 16S rRNA or -tubulin genes and were not derived from contaminants. Differential staining with monoclonal antibody as described below also was observed. Consequently, the methods development work reported below was performed using infectious, large subpopulation spores.
Development/Optimization of Continuous Flow Centrifugation Methods
Several of the logistic and procedural aspects involved in concentrating microsporidia by continuous flow centrifugation were completed during this project period and are described below.
A blood cell separator was used to concentrate microsporidia from filtered tap water. Before centrifuging a sample, the plastic separation chamber and input/output tubing were primed with phosphate buffered saline (PBS) containing 0.1 percent Tween 80. Studies have shown that coating these plastic surfaces with surfactant enhances removal of the collected particles after centrifugation. Ten liters filtered tap water was spiked with E. intestinalis spores that had been prestained with FITC-conjugated anti-microsporidia antibody. These samples were processed at a feed rate of 70 mL/minute and a relative gravitational force of approximately 900 x g. Microsporidia were retained in the separation chamber, and the supernatant was pushed out of the bag via the output line. After centrifugation, the contents of the separation chamber were drained into a beaker. The separation chamber was rinsed multiple times with a PBS-Tween 80 solution to dislodge any spores that may have adhered to the inner chamber walls. Rinse volumes and separation chamber contents were poured into four 50 mL polycarbonate tubes and centrifuged at 31,500 x g for 50 minutes. Multiple rinses and high speed centrifugations were performed to concentrate the original 10 L sample to a final volume of 400 µL. This volume was transferred to treated well slides and allowed to dry completely. A drop of mounting media was added and a coverslip was placed on top of the well slide. Each well was scanned at 200x magnification with a Nikon Optiphot epifluorescent microscope using a FITC (Ex 450-490, DM 505, BA 520) filter cube. Spore identity was confirmed 400x magnification. Microsporidia were identified based on the apple-green fluorescence of FITC and the characteristic size and morphology of the spores.
The effectiveness of the continuous flow centrifugation procedure in recovering spores is given by percent recovery (i.e., total number of spores added to the water sample divided by the number recovered multiplied by 100). Three preliminary trials with pre-stained microsporidia seeded into 10 L of filtered tap water have been completed. Recoveries ranged from 82 percent to 89 percent. Additional trials will be performed that will split the final 400 µL concentrate into two aliquots: one to be counted by flow cytometry and the other to be counted by epifluorescence microscopy.
Sample Concentration/Purification By Flow Cytometry
Several antibodies were evaluated for use in concentration/purification methods. Monoclonal antibody 7G7 (Ted Nash, NIH) was conjugated to FITC and used to stain freshly harvested spore suspensions. This antibody exhibited a variable staining pattern, brightly staining the small spore subpopulation but only dimly staining the large spore subpopulation. Rabbit polyclonal antibody produced against formalin-treated Encephalitozoon spores stained only E. cuniculi. An additional rabbit polyclonal generated against UV-treated Encephalitozoon spores was shown to be highly reactive with all three Encephalitozoon species. Flow cytometry was evaluated for the ability to recover microsporidia from sample concentrates. Suspensions containing either 100 spores prelabeled with FITC-conjugated rabbit anti- Encephalitozoon polyclonal antibody or 100 unlabeled spores were prepared using established flow cytometric spiking methods. Those suspensions containing prelabeled spores were flow-sorted onto three well microscope slides using the spores' physical and fluorescence properties and examined with epifluorescence microscopy, whereas suspensions containing unlabeled spores were stained with a FITC-conjugated polyclonal antibody prior to recovery experiments. Recovery of prelabeled spores averaged 93.1 percent, sd=4.2, n=10; while recoveries of spores labeled in solution averaged 82.2 percent, sd=3.3, n=10.
Diagnostic Assay/Viability Testing
RT-PCRCC assays were performed on Encephalitozoon intestinalis spores using primers designed to the 16S rRNA or -tubulin genes to determine if viable spores could be differentiated from non-viable spores. In addition, the assays were performed with E. cuniculi and E. hellem spores to test whether these primer pairs could be used to detect multiple Encephalitozoon species. Microsporidial spores were heat inactivated and viability was determined by both RT-PCRCC assays and standard in vitro cell culture coverslip assays, performed in tandem. Viability, as measured by the RT-PCRCC assays using the -tubulin specific primers, compared favorably with a standard cell culture assay. In contrast, the 16S rRNA-specific primers were able to amplify non-viable spores, limiting their usefulness for determination of viability. Sensitivity determinations of the RT-PCRCC assays for the three different primer pairs and the detection of microsporidial spores in concentrated water pellets currently are under investigation.
PCR assays using primers designed to 16S rRNA were performed on E. intestinalis, E. cuniculi, and E. hellem spores and were able to amplify all three species. Thus, PCR may be used as a detection method and the RT-PCTCC assay as a viability assay.
Cell Culture Infectivity/Dose Response
The TCID50 was determined using a logit dose response model. Briefly, the response logit (RL) was calculated for each dose of spores as the natural logarithm (ln) of the proportion of infected tissue culture wells (P) divided by one minus the proportion of inoculated wells [RL = ln(P/(1-P)]. The response logit values were treated as the dependent variables for regression analysis with the logarithm base ten (log10) of the number of spores in each dose serving as the independent (x) variable. Regression analysis then was used to perform a least squares regression to calculate the regression model parameters. The logit dose response regression model was used to determine the TCID50 for each species. The TCID50 for E. cuniculi (logit dose response equation y = 1.4878x-1.7756) was calculated to be 16 spores, for E. hellem (logit dose response equation y = 2.521x-4.974) it was 94 spores, and for E. intestinalis (logit dose response equation y = 2.1609x-3.1053) it was 27 spores.
Future Activities:
During the next reporting period, the investigators will continue to evaluate the ability of CFC to recover microsporidia from spiked tap and natural waters. Immunomagnetic separation methods using biotinylated antibodies will be pursued for use with highly turbid environmental samples. Flow cytometry parameters will continue to be evaluated/optimized using CFC-generated concentrates that have been spiked with labeled and unlabeled E. intestinalis spores.
In the next project period, the investigators also will perform RT-PCR CC assays using primers designed to the 70HSP genes and compare these results with the 16S rRNA or -tubulin genes to determine if viable spores could be differentiated from non-viable spores. Primers will be tested with all three Encephalitozoon species. PCR studies using the three primer sets listed above also will be performed to determine the sensitivity of PCR assays for potential detection assays on water pellets.
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.